Compositions and methods of use of phorbol esters

ABSTRACT

Methods and compositions containing a phorbol ester or a derivative of a phorbol ester are provided for the treatment of cytopathic diseases. Cytopathic diseases may be caused by a variety means such as viral infections like HIV and AIDS in a mammalian subject. The methods and compositions of the invention are effective for inhibiting de novo HIV infection, upregulating viral expression from latent provirus, inhibiting HIV-induced cytopathic effects, down regulating the HIV receptor, increasing ThI cytokine expression, and decreasing Th2 cytokine expression. Additional compositions and methods are provided which employ a phorbol ester or derivative compound in combination with at least one additional agent such as those used in HAART protocols or therapeutic agents used to treat opportunistic infections due to HIV in mammalian subjects.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/794,467, filed Mar. 11, 2013 (now allowed), which is a Continuationof U.S. application Ser. No. 13/595,072, filed Aug. 27, 2012 (nowabandoned), which is a Continuation of U.S. application Ser. No.12/023,753, filed Jan. 31, 2008 (now abandoned), which claims benefit ofU.S. Provisional Application No. 60/898,810, filed Jan. 31, 2007; thedisclosures of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates generally to the field of cytopathicdiseases. More specifically, the invention relates to compositionscontaining and methods of using phorbol esters to treat cytopathicconditions and diseases that cause such cytopathic conditions.

BACKGROUND

Phorbol is a natural, plant-derived organic compound of the tiglianefamily of diterpenes. It was first isolate in 1934 as a hydrolysisproduct of croton oil derived from the seeds of Croton tiglium, a leafyshrub of the Euphorbiaceae family that is native to Southeastern Asia.Various esters of phorbol have important biological properties includingthe reported ability to mimic diacylglycerols and activate proteinkinase C (PKC), modulating downstream cell signaling pathways includingthe mitogen-activated protein kinase (MAPK) pathways. Phorbol esters areadditionally thought to bind to chimaerins, the Ras activator RasGRP,and the vesicle-priming protein Munc-13 (Brose N, Rosenmund C., JCellSci; 115:4399-411 (2002)). Some phorbol esters also induce nuclearfactor-kappa B (NF-κB). The most notable physiological property ofphorbol esters is their reported capacity to act as tumor promoters.

12-O-tetradecanoylphorbol-13-acetate (TPA), also calledphorbol-12-myristate-13-acetate (PMA), is a phorbol ester used in modelsof carcinogenesis as an inducer for differentiation and/or apoptosis inmultiple cell lines and primary cells. TPA has also been reported tocause an increase in circulating white blood cells and neutrophils inpatients whose bone marrow function has been depressed by chemotherapy.(Han Z. T. et al. Proc. Natl. Acad. Sci. 95,5363-5365 (1998)) andinhibit the HIV-cytopathic effects on MT-4 cells. (Mekkawy S. et al.,Phytochemistry 53,47-464 (2000)). However, due to a variety of factors,including caustic reactions when contacted with the skin and concernsfor its potential toxicity, TPA has not been shown to be an effectivetool for treating, managing, or preventing HIV or AIDS.

Current therapeutics for cytopathic diseases such as various forms ofneoplastic disease and viral diseases such as HIV and AIDS suffer from anumber of drawbacks such as insufficient potency and intolerable sideeffects. For many patients, toxic side effects of diminish their qualityof life to such an extent they simply stop taking their medications. Forothers, therapeutic schedules are so complicated and inconvenient thatcompliance is limited. Other patients experience excellent resultsinitially, but suffer relapses despite full compliance with therapeuticregimens.

Treatment failure in most HIV cases is attributed to the emergence ofresistant strains of HIV. Incomplete viral suppression caused byinsufficient drug potency, poor compliance due to complicated drugregimens, and other factors contribute to this problem. Additionally,during the long period of clinical latency of HIV infection, a subset ofquiescent memory CD4 T-cells harbor integrated but transcriptionallysilent proviruses. This reservoir protects latent HIV from retroviraltherapy and poses a substantial barrier to eradication of HIV ininfected patients.

Cancer treatments generally involve a combination of surgery,chemotherapy, hormonal therapy and/or radiation treatment to eradicateneoplastic cells in a patient. However, all of these approaches posesignificant drawbacks and added risks such as increased susceptibilityto infection. Surgery, for example, may be contraindicated due to thehealth of a patient. Additionally, it may be difficult to obtain clearmargins around a rumor, resulting in some neoplastic tissue being leftbehind and an increased chance of recurrence of the disease. Almost allcurrent chemotherapeutic agents are toxic, and chemotherapy causessignificant side effects including severe nausea, bone marrowdepression, and immunosuppression. They also cannot be specificallytargeted to cancer cells and therefore may kill healthy cells as well ascancerous ones. Additionally, there are frequently relapsed/refractoryneoplasms which are resistant to current therapeutics.

There is clearly a need for new and more effective treatments forindividuals suffering from cytopathic disorders, including those causedby neoplastic disease as well as viral infections such as HIV and AIDS.

SUMMARY OF THE EXEMPLARY EMBODIMENTS OF THE INVENTION

The present invention relates to compositions containing and methods ofusing phorbol esters in the treatment of cytopathic diseases.

In one embodiment, phorbol esters and derivatives of phorbol esters areused to treat cytopathic diseases such as HIV and associated conditionssuch as AIDS. The compositions and methods of the present invention mayaccomplish the treatment of HIV and associated conditions such as AIDSby any means possible. In some embodiments, the compositions and methodsmay modify HIV receptor activity in mammalian subjects. In anotherembodiment, compositions and methods may decrease the number of latentHIV reservoirs in an HIV-infected subject. In a further embodiment, itmay enhance HIV activation in latent pro-viral cells. In additionalembodiments, it may inhibit HW-cytopathic effects.

In another embodiment, compositions containing phorbol esters andphorbol ester derivatives may be used for treating and managing symptomsof HIV and AIDS in mammalian subjects. Targeted symptoms for treatmentand management employing the compositions and methods of the inventionsinclude, but are not limited to, oral lesions, fatigue, skin thrush,fever, lack of appetite, diarrhea, apthous ulcers, malabsorption,thrombocytopenia, weight loss, anemia, lymph node enlargement,susceptibility to and severity of secondary conditions such asmycobacterium avium complex, salmonellosis, syphilis, neuroshyphilis,turberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis,coccidioidomycosis, listeriosis, pelvic inflammatory disease, Burkitt'slymphoma, cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma,lymphoma, systemic non-Hodgkin's lymphoma (NHL), primary CNS lymphoma,cryptosporidiosis, isosporiasis, microsporidiosis, pneumocystis cariniipneumonia (PCP), toxoplasmosis, cytomegalovirus (CMV), hepatitis, herpessimplex, herpes zoster, human papiloma virus (HPV, genital warts,cervical cancer), molluscum contagiosum, oral hairy leukoplakia (OHL),and progressive multifocal leukoencephalopathy (PML).

In a further embodiment, compounds containing phorbol esters andderivatives of phorbol esters may be used to treat cytopathic conditionssuch as neoplastic diseases. Such neoplasms may be malignant or benign.In some embodiments, neoplasms may be solid or non-solid cancers. Inother embodiments, the neoplasms may be relapses. In another embodiment,the neoplasms may be refractory. Exemplary neoplasms include, but arenot limited to, hematologic malignancies/bone marrow disorders,including, but not limited to, leukemia, including acute myeloidleukemia (AML), chronic myeloid leukemia (CML), chronic myeloid leukemiablast crisis, myelodysplasia, and myeloproliferative syndrome; lymphoma,including Hodgkins and non-Hodgkins lymphoma; subcutaneousadenocarcinoma; ovarian teratocarcinoma; and prostate cancer. Otherneoplastic conditions amenable to treatment using the methods andcompositions include other cancer disorders and conditions, includingsolid tumors of various types, where successful treatment and/orremission will be determined according to conventional methods, such asdetermining size reduction of solid tumors, and/or histopathologicalstudies to assess growth, stage, metastatic state or potential, presenceor expression levels of histological cancer markers, etc.

Compositions and methods herein may additionally be used to treatsymptoms of neoplastic disease including, but not limited to, anemia;chronic fatigue; excessive or easy bleeding, such as bleeding of thenose, gums, and under the skin; easy bruising, particularly bruisingwith no apparent cause; shortness of breath; petechiae; recurrent fever;swollen gums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and weakness.

In yet another embodiment, the phorbol esters and derivatives of phorbolesters may be used to modulate cell signaling pathways. Such modulationmay have a variety of results, for example, in some embodiments, the useof compositions containing phorbol esters and derivatives of phorbolesters may increase white blood cell counts in mammalian subjects. Inanother embodiment, compositions containing phorbol esters and/orphorbol ester derivatives may alter the release of Th1 cytokines inmammalian subjects. In a further embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the release ofinterleukin 2 (IL-2) in mammalian subjects. In an additional embodiment,compositions containing phorbol esters and/or phorbol ester derivativesmay alter the release of interferon in mammalian subjects. In yetanother embodiment, compositions containing phorbol esters and/orphorbol ester derivatives may alter the rate of ERK phosphorylation.

The invention achieves the foregoing and satisfies additional objectsand advantages by providing novel and surprisingly effective methods andcompositions for modulating cell signaling pathways and/or treatingcytopathic diseases and symptoms of cytopathic diseases or conditionsusing compositions containing a phorbol ester or derivative compositionof the Formula I, below:

wherein R₁ and R₂ may be hydrogen;

wherein the alkyl group contains 1 to 15 carbon atoms;

and substituted derivatives thereof and R₃ may be hydrogen or

and substituted derivatives thereof.

In another embodiment, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof. In yet another embodiment, eitherR₁ or R₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino; and/or similar type radicals.

In a further embodiment, the invention achieves these objects andsatisfies additional objects and advantages by providing novel andsurprisingly effective methods and compositions for modulating cellsignaling pathways and/or treating cytopathic diseases or conditionsassociated with cytopathic diseases using an exemplary phorbol estercomposition such as 12-O-tetradecanoylphorbol-13-acetate (TPA) ofFormula II, below:

Useful phorbol esters and related compounds and derivatives within theformulations and methods of the invention include, but are not limitedto, other pharmaceutically acceptable active salts of said compounds, aswell as active isomers, enantiomers, polymorphs, glycosylatedderivatives, solvates, hydrates, and/or prodrugs of said compounds.Exemplary forms of phorbol esters for use within the compositions andmethods of the invention include, but are not limited to, phorbol13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate; phorbol12,13-diacetate; phorbol 13,20-diacetate; phorbol 12,13-dibenzoate;phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate; phorbol12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol 12-myristate;phorbol 13-myristate; phorbol 12-myristate-13-acetate (also known as TPAor PMA); phorbol 12,13,20-triacetate; 12-deoxyphorbol 13-angelate;12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol 13-isobutyrate;12-deoxyphorbol 13-isobutyrate-20-acetate; 12-deoxyphorbol13-phenylacetate; 12-deoxyphorbol 13-phenylacetate 20-acetate;12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate 13-decanoate;12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate.

In exemplary embodiments, the compositions and methods of the inventionemploy a phorbol ester compound of Formula I to treat and/or preventsymptoms of cytopathic diseases including, but not limited to, symptomsof HIV and AIDS or other diseases and conditions associated with HIV andAIDS such as opportunistic infections, as well as symptoms of neoplasticdiseases or other diseases and conditions associated with neoplasticdiseases.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI, particularly TPA, according to the methods of the invention include,but are not limited to, subjects with HIV and AIDS, as well as subjectswith symptoms, or secondary or opportunistic diseases associated withHIV and AIDS, such as oral lesions, fatigue, skin thrush, fever, lack ofappetite, diarrhea, apthous ulcers, malabsorption, thrombocytopenia,weight loss, anemia, lymph node enlargement, mycobacterium aviumcomplex, salmonellosis, syphilis, neuroshyphilis, turberculosis (TB),bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis,listeriosis, pelvic inflammatory disease, Burkitt's lymphoma,cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma, lymphoma,systemic non-Hodgkin's lymphoma (NHL), primary CNS lymphoma,cryptosporidiosis, isosporiasis, microsporidiosis, pneumocystis cariniipneumonia (PCP), toxoplasmosis, cytomegalovirus (CMV), hepatitis, herpessimplex, herpes zoster, human papiloma virus (HPV, genital warts,cervical cancer), molluscum contagiosum, oral hairy leukoplakia (OHL),and progressive multifocal leukoencephalopathy (PML).

Additional mammalian subjects amenable to treatment with phorbol estersof Formula I, particularly TPA, according to the methods of the presentinvention include, but are not limited to, subjects suffering fromneoplastic diseases including malignant neoplastic diseases such assolid and non-solid cancers. Non-solid cancers may include, hematologicmalignancies/bone marrow disorders, including, but not limited to,leukemia, including acute myeloid leukemia (AML), chronic myeloidleukemia (CML), chronic myeloid leukemia blast crisis, myelodysplasia,myeloproliferative syndrome. Solid cancers may include, but are notlimited to, lymphoma, including Hodgkins and non-Hodgkins lymphoma,subcutaneous adenocarcinoma, ovarian teratocarcinoma, and prostatecancer. Subjects amenable to treatment with phorbol esters of Formula I,particularly TPA additionally include those suffering from symptoms ofsuch neoplastic diseases such as, but not limited to, anemia; chronicfatigue; excessive or easy bleeding, such as bleeding of the nose, gums,and under the skin; easy bruising, particularly bruising with noapparent cause; shortness of breath; petechiae; recurrent fever; swollengums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and weakness. In some embodiments, such cancers may be relapsesor refractory.

These and other subjects are effectively treated, prophylacticallyand/or therapeutically, by administering to the subject and effectiveamount of a phorbol ester of Formula I sufficient to prevent or reduceviral load, decrease latent reservoirs of HIV, increase immuneresponsiveness, increase the release of Th1 cytokines, prevent or reducesymptoms and conditions associated with HIV and AIDS, decrease and/oreliminate neoplastic cells, increase white blood cell counts, induceremission, maintain remission, prevent or reduce symptoms and conditionsassociated with malignancies and/or increase ERK phosphorylation.Therapeutically useful methods and formulations of the invention willeffectively use a phorbol ester of Formula I in a variety of forms, asnoted above, including any active, pharmaceutically acceptable salts ofsaid compounds, as well as active isomers, enantiomers, polymorphs,solvates, hydrates, prodrugs, and/or combinations thereof. TPA offormula II is employed as an illustrative embodiment of the inventionwithin the examples herein below.

Within additional aspects of the invention, combinatorial formulationsand methods are provided which employ an effective amount of a phorbolester of Formula I in combination with one or more secondary oradjunctive active agent(s) that is/are combinatorially formulated orcoordinately administered with the phorbol ester compound of Formula Ito yield an effective response in the subject. Exemplary combinatorialformulations and coordinate treatment methods in the treatment of viralcytopathic diseases such as HIV and AIDS employ the phorbol estercompound of Formula I in combination with one or more additional,retroviral, HIV or AIDS treating or other indicated secondary oradjunctive therapeutic agents. Such combinatorial formulations andcoordinate treatment methods may, for example, follow or be derived fromvarious highly active antiretroviral therapy protocols (HAART protocols)and include regimens such as, but not limited to, two nucleosideanalogue reverse transcriptase inhibitor plus one or more proteaseinhibitor or non-nucleoside analogue reverse transcriptase inhibitoramong other combinations. Other combinatorial formulations andcoordinate treatment methods may, for example, include treatments foropportunistic infections as well as the compounds for the HAARTprotocols. The secondary or adjunctive therapeutic agents used incombination with, e.g., TPA, in these embodiments may possess direct orindirect antiviral effects, alone or in combination with, e.g. TPA, mayexhibit other useful adjunctive therapeutic activity in combinationwith, e.g. TPA (such as HIV preventing, HIV treating, HIV reservoiractivating, Th1 cytokine increasing activity); or may exhibit adjunctivetherapeutic activity useful for treating opportunistic infectionsassociated with HIV alone or in combination with, e.g. TPA

Useful adjunctive therapeutic agents in these combinatorial formulationsand coordinate treatment methods include, for example, proteaseinhibitors, including, but not limited to, saquinavir, indinavir,ritonavir, nelfinavir, atazanavir, darunavir, fosamprenavir, tipranavirand amprenavir; nucleoside reverse transcriptase inhibitors includingbut not limited to, zidovudine, didanosine, stavudine, lamivudine,zalcitabine, emtricitabine, tenofovir disoproxil fumarate, AVX754 andabacavir; non-nucleoside reverse transcriptase inhibitors including, butnot limited to, nevaripine, delavirdine, calanolide A, TMC125 andefavirenz; combination drugs including, but not limited to,efavirenz/emtricitabine/tenofovir disoproxil fumarate,lamivudine/zidovudine, abacavir/lamivudine,abacavir/lamivudine/zidovudine, emtricitabine/tenoforvir disoproxilfumarate, sulfamethoxazole/trimethoprim, and lopinavir/ritonavir; entryand fusion inhibitors, including, but not limited to, enfuvirtide,AMD070, BMS-488043, fozivudine tidoxil, GSK-873,140, PRO 140, PRO 542,Peptide T, SCH-D, TNX-355, and UK-427, 857; treatments for opportunisticinfections and other conditions associated with AIDS and HIV including,but not limited to, acyclovir, adefovir dipivoxil, aldesleukin,amphotericin b, azithromycin, calcium hydroxylapatite, clarithromycin,doxorubicin, dronabinol, entecavir, epoetin alfa, etoposide,fluconazole, ganciclovir, immunoglobulins, interferon alfa-2, isoniazid,itraconazole, megestrol, paclitaxel, peginterferon alfa-2, pentamidine,poly-1-lactic acid, ribavirin, rifabutin, rifampin, somatropin,testosterone, trimetrexate, and valganciclovir; integrase inhibitorsincluding, but not limited to, GS 9137, MK-0518; microbicides,including, but not limited to, BMS-378806, C31G, carbopol 974P,carrageenan, cellulose sulfate, cyanovirin-N, dextran sulfate,hydroxyethyl cellulose, PRO 2000, SPL7013, tenofovir, UC-781 and IL-2.

Exemplary combinatorial formulations and coordinate treatment methods inthe treatment of neoplastic disease employ the phorbol ester compound ofFormula I in combination with one or more additional, neoplastic diseasetreating or other indicated, secondary or adjunctive therapeutic agents.The secondary or adjunctive therapeutic agents used in combination with,e.g., TPA, in these embodiments may possess direct or indirectchemotherapeutic effects, alone or in combination with, e.g. TPA, mayexhibit other useful adjunctive therapeutic activity in combinationwith, e.g. TPA (such as cytotoxic, anti-inflammatory, NF-κB inhibiting,apoptosis inducing, Th1 cytokine increasing activity); or may exhibitadjunctive therapeutic activity useful for treating neoplasms orassociated symptoms alone or in combination with, e.g. TPA.

Useful adjunctive or secondary therapeutic agents in these combinatorialformulations and coordinate treatment methods include doxorubicin,vitamin D3, cytarabine, cytosine arabinoside, daunorubicin,cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine,mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin,etoposide phosphate, fludarabine, methotrexate, etoposide,dexamethasone, and choline magnesium trisalicylate. In addition,adjunctive or secondary therapies may be used such as, but not limitedto, radiation treatment, hormone therapy and surgery.

The forgoing and additional objects, features, aspects and advantages ofthe present invention will become apparent from the following detaileddescription.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

Novel methods and compositions have been identified for use inpreventing and/or treating cytopathic diseases and conditions inmammalian subjects. In various embodiments, the methods and compositionsare effective to prevent or treat HIV and AIDS and related conditions,diseases caused by HIV and AIDS, and/or diseases acquired because of HIVor AIDS infection. In other embodiments, the methods and compositionsare effective to prevent or treat neoplastic diseases and symptoms ofsuch diseases. Such neoplastic diseases may or may not be malignant. Insome embodiments, the neoplastic diseases may be solid or non-solidcancers. In other embodiments, the cancers may be refractory orrelapses.

Formulations and methods provided herein employ a phorbol ester orderivative compound of Formula I, above, including all activepharmaceutically acceptable compounds of this description as well asvarious foreseen and readily provided complexes, salts, solvates,isomers, enantiomers, polymorphs and prodrugs of these compounds andcombinations thereof as novel HIV and AIDS treating compounds.

Formulations and methods provided herein additionally employ a phorbolester or derivative compound of Formula I, above, including all activepharmaceutically acceptable compounds of this description as well asvarious foreseen and readily provided complexes, salts, solvates,isomers, enantiomers, polymorphs and prodrugs of these compounds andcombinations thereof in the treatment of neoplastic diseases.

Viral load decreasing formulations and methods provided herein employ aphorbol ester or derivative compound of Formula I, above, including allactive pharmaceutically acceptable compounds of this description as wellas various foreseen and readily provided complexes, salts, solvates,isomers, enantiomers, polymorphs and prodrugs of these compounds andcombinations thereof as novel viral load decreasing agents.

Apoptosis inducing formulations and methods provided herein employ aphorbol ester or derivative of compound of Formula I, above, includingall active pharmaceutically acceptable compounds of this description aswell as various foreseen and readily provided complexes, salts,solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as chemotherapeutic agents thatinduce apoptosis in neoplasms.

Remission inducing formulations and methods provided herein employ aphorbol ester or derivative of compound of Formula I, above, includingall active pharmaceutically acceptable compounds of this description aswell as various foreseen and readily provided complexes, salts,solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as anti-neoplasm agents.

Immune responsiveness increasing formulations and methods providedherein employ a phorbol ester or derivative of compound of Formula I,above, including all active pharmaceutically acceptable compounds ofthis description as well as various foreseen and readily providedcomplexes, salts, solvates, isomers, enantiomers, polymorphs andprodrugs of these compounds and combinations thereof as immunestimulatory compounds.

Th1 cytokine increasing formulations and methods provided herein employa phorbol ester or derivative of compound of Formula I, above, includingall active pharmaceutically acceptable compounds of this description aswell as various foreseen and readily provided complexes, salts,solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as novel Th1 cytokine increasingagents.

A broad range of mammalian subjects, including human subjects, areamenable to treatment using the formulations and methods of theinvention. These subjects include, but are not limited to, individualssuffering from cytopathic diseases or conditions including neoplasticdiseases and viral cytopathic diseases such as HIV and AIDS.

Subjects amenable to treatment include HIV+ human and other mammaliansubjects presenting with oral lesions, fatigue, skin thrush, fever, lackof appetite, diarrhea, apthous ulcers, malabsorption, thrombocytopenia,weight loss, anemia, lymph node enlargement, susceptibility to andseverity of secondary conditions such as mycobacterium avium complex,salmonellosis, syphilis, neuroshyphilis, turberculosis (TB), bacillaryangiomatosis, aspergillosis, candidiasis, coccidioidomycosis,listeriosis, pelvic inflammatory disease, Burkitt's lymphoma,cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma, lymphoma,systemic non-Hodgkin's lymphoma (NHL), primary CNS lymphoma,cryptosporidiosis, isosporiasis, microsporidiosis, pneumocystis cariniipneumonia (PCP), toxoplasmosis, cytomegalovirus (CMV), hepatitis, herpessimplex, herpes zoster, human papiloma virus (HPV, genital warts,cervical cancer), molluscum contagiosum, oral hairy leukoplakia (OHL),and progressive multifocal leukoencephalopathy (PML).

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I is disclosed herein is/areeffectively formulated or administered as an agent effective fortreating HIV/AIDS and/or related disorders. In exemplary embodiments,TPA is demonstrated for illustrative purposes to be an effective agentin pharmaceutical formulations and therapeutic methods, alone or incombination with one or more adjunctive therapeutic agent(s). Thepresent disclosure further provides additional pharmaceuticallyacceptable phorbol ester compounds in the form of a native or syntheticcompound, including complexes, derivatives, salts, solvates, isomers,enantiomers, polymorphs and prodrugs of the compounds disclosed herein,and combinations thereof, which are effective as therapeutic agentswithin the methods and compositions of the invention in the treatment ofHIV/AIDS and related conditions.

Acquired immune deficiency syndrome or acquired immunodeficiencysyndrome (AIDS or Aids) is a collection of symptoms and infectionsresulting from damage to the immune system caused by infection with thehuman immunodeficiency virus (HIV). The damage to the immune systemleaves individuals prone to opportunistic infections and tumors.Although treatments for AIDS and HIV exist to slow the virus'sprogression and the severity of the symptoms, there is no known cure.

HIV is a retrovirus that primarily infects components of the humanimmune system such as CD4+ T cells, macrophages and dendritic cells.When CD4+ T cells are destroyed and their total count decreases to below200 CD4+ T cells/μL of blood or the percentage of CD4+ T-cell as afraction of the total lymphocytes falls to less than 14%, cellularimmunity is lost, leading to AIDS.

It is currently believed that a change in the T_(h)1 and T_(h)2 cytokinebalance can contribute to immune dysregulation associated with HIVinfection. T_(h)1 cells produce cytokines that stimulate proliferationof cytotoxic T cells. T_(h)2 cells produce cytokines that areresponsible for activation of the humoral immune responses in healthypeople. Progression from HIV infection to AIDS is characterized by adecrease in levels of T_(h)1 cytokines IL-2IL-12 and IFN-γ with aconcomitant increase in levels of T_(h)2 cytokines IL-4IL-5 and IL-10.(Clerci, Immunology Today, v. 14, No. 3, p. 107-110, 1993; Becker, VirusGenes 28:1, 5-18 (2004)). Resistance to HIV infection and/or resistanceto progression to AIDS may therefore be dependent on a T_(h)1>T_(h)2dominance.

A fraction of CD4+ memory T cells contain integrated transcriptionallyinactive proviruses for HIV. These latent reservoirs may be activated toproduce active infectious virus following activation by specificantigens or cytokines. The half life of these CD4 memory T cells is atleast 44 months making it extremely difficult to eliminate HIV andrequiring extended continuation of antiretroviral therapy even when HIVlevels in the peripheral blood are undetectable.

Prostratin, 12-deoxyphorbol 13-acetate, a non-tumor promoting phorbolester, has reportedly shown some effectiveness for inhibiting HIVinduced cell killing and viral replication. Prostratin reportedlyactivated viral expression in latently-infected cell lines, but hadlittle or no effect on chronically-infected cell lines. (Gulakowski, etal., Antiviral Research v. 33, 87-97 (1997); Williams, et al., JBC v.279, No. 40, P. 42008-42017 (2004)). Protratin represents a distinctsubclass of protein kinase C activators which has unique biologicalactivities that differ from tumor-promoting phorbol esters such as TPA.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI, particularly TPA, according to the methods of the present inventionadditionally include, but are not limited to, mammalian subjects withneoplastic diseases including solid and non-solid cancers, includinghematologic malignancies/bone marrow disorders, such as leukemia,including acute myeloid leukemia (AML), chronic myeloid leukemia (CML),chronic myeloid leukemia blast crisis, myelodysplasia,myeloproliferative syndrome; lymphoma, including Hodgkins andnon-Hodgkins lymphoma; subcutaneous adenocarcinoma; ovarianteratocarcinoma; and prostate cancer. In some embodiments, such cancersmay be relapses or refractory.

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I as disclosed herein is/areeffectively formulated or administered as an agent effective fortreating neoplastic diseases. In exemplary embodiments, TPA isdemonstrated for illustrative purposes to be an effective agent inpharmaceutical formulations and therapeutic methods, alone or incombination with one or more adjunctive therapeutic agent(s). Thepresent disclosure further provides additional, pharmaceuticallyacceptable phorbol ester compounds in the form of a native or syntheticcompound, including complexes, derivatives, salts, solvates, isomers,enantiomers, polymorphs and prodrugs of the compounds disclosed herein,and combinations thereof, which are effective as therapeutic agentswithin the methods and compositions of the invention in the treatment ofneoplastic diseases and symptoms of such diseases.

Neoplastic disease is any growth or tumor caused by abnormal anduncontrolled cell division; it may spread to other parts of the bodythrough the lymphatic system or the blood stream. Such growths may bemalignant or benign, solid or non-solid.

In some embodiments, the neoplastic diseases may be a hematologicalneoplasm/bone marrow disorder such as acute myeloid leukemia (AML). AML(also called acute myelogenous leukemia, acute myeloblastic leukemia,acute granulocytic leukemia, and acute nonlymphocytic leukemia) is themost common type of acute leukemia in adults. In AML, stem cellsproduced by the bone marrow usually develop into a type of immaturewhite blood cell called myeloblasts (or myeloid blasts). In individualssuffering from AML, these myeloblasts do not mature into healthy whiteblood cells. Additionally, stem cells in individuals with AML maydevelop into abnormal red blood cells or platelets. The lack of normalblood cells increases incidences of infection, anemia, and easybleeding. Additionally, the leukemia cells can spread outside the bloodto other parts of the body, including the central nervous system (brainand spinal cord), skin, and gums.

The average age of a patient with AML is over 64 years of age. Patientsover the age of 60 treated for AML with standard chemotherapeutics havea remission rate of less than 20%. Additionally, patients who developAML after an antecedent hematologic disorder or prior leukemogenicchemotherapy/radiation therapy have similarly poor outcomes.

Phorbol is a natural, plant-derived polycyclic alcohol of the tiglianefamily of diterpenes. It was first isolated in 1934 as the hydrolysisproduct of croton oil derived from the seeds of Croton tiglium. It iswell soluble in most polar organic solvents and in water. Esters ofphorbol have the general structure of Formula I, below:

wherein R₁ and R₂ are selected from the group consisting of hydrogen;

wherein the alkyl group contains 1 to 15 carbon atoms,

and substituted derivatives thereof and R₃ may be hydrogen,

or substituted derivatives thereof.

The term “lower alkyl” or “lower alkenyl” as used herein means moietiescontaining 1-7 carbon atoms. In the compounds of the Formula I, thealkyl or alkenyl groups may be straight or branched chain. In someembodiments, either or both R₁ or R₂, are long chain carbon moiety(i.e., Formula I is decanoate or myristate).

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino and similar type radicals.

Organic and synthetic forms of phorbol esters, including anypreparations or extracts from herbal sources such as croton tiglium, arecontemplated as useful compositions comprising phorbol esters (orphorbol ester analogs, related compounds and/or derivatives) for usewithin the embodiments herein. Useful phorbol esters and/or relatedcompounds for use within the embodiments herein will typically have astructure as illustrated in Formula I, although functionally equivalentanalogs, complexes, conjugates, and derivatives of such compounds willalso be appreciated by those skilled in the art as within the scope ofthe invention.

In more detailed embodiments, illustrative structural modificationsaccording to Formula I above will be selected to provide usefulcandidate compounds for treating and/or preventing HIV and AIDS and/orneoplastic diseases, wherein: at least one of R₁ and R₂ are other thanhydrogen and R₃ is selected from the group consisting of hydrogen,

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.

An exemplary embodiment of a phorbol ester compound of Formula I usefulin the treatment of cytopathic diseases such as HIV and AIDS and/orneoplastic diseases, particularly AML, is found in phorbol12-myristate-13-acetate (also known as PMA or12-O-tetradecanoyl-phorbol-13-acetate (TPA)) shown in Formula II, below.

Additional useful phorbol esters and related compound and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Further exemplary forms of phorbol esters for use within thecompositions and methods of the invention include, but are not limitedto, phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate;phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate;12-deoxyphorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate;12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate;12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol13-acetate; phorbol 12-acetate; and phorbol 13-acetate.

Cytopathic disease treating compositions herein comprise HIV- andAIDS-treating compositions comprising an anti-AIDS effective amount of aphorbol ester compound of Formula I, which is effective for prophylaxisand/or treatment of HIV, AIDS, and/or HIV-related symptoms, includingopportunistic infections, in a mammalian subject. An “anti-HIV”,“anti-AIDS”, or “AIDS treating” effective amount of the active compoundis therapeutically effective, in a single or multiple unit dosage form,over a specified period of therapeutic intervention, to measurablyalleviate one or more symptoms of AIDS in a subject, and/or to alleviateone or more symptom(s) or condition(s) associated with HIV infection inthe subject. Within exemplary embodiments, the compositions of theinvention are effective in treatment methods to alleviate symptoms ofAIDS or other HIV-related conditions in human and other mammaliansubjects vulnerable to HIV infection.

Cytopathic disease treating compositions herein additionally maycomprise chemotherapeutic compositions comprising an anti-neoplasticeffective amount of a phorbol ester or derivative compound of Formula I,which is effective for maintenance and treatment of malignancies orsymptoms caused by cancer in a mammalian subject. A “chemotherapeutic”,“anti-tumor,” “cancer treating”, “apoptosis inducing”, “remissioninducing”, “remission maintaining” effective amount of the activecompound is therapeutically effective, in a single or multiple unitdosage form, over a specified period of therapeutic intervention, tomeasurably alleviate one or more symptoms of malignancy in a subject,and/or to alleviate one or more symptom(s) or condition(s) associatedwith malignancy in the subject. Within exemplary embodiments, thecompositions of the invention are effective in treatment methods toalleviate symptoms of neoplastic disease related conditions in human andother mammalian subjects vulnerable to malignancies.

Cytopathic disease treating, including chemotherapeutic and HIVtreating, compositions of the invention typically comprise an effectiveamount or unit dosage of a phorbol ester compound of Formula I, whichmay be formulated with one or more pharmaceutically acceptable carriers,excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers,and/or other additives that may enhance stability, delivery, absorption,half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduceadverse side effects, or provide other advantages for pharmaceuticaluse. Effective amounts of a phorbol ester compound or related orderivative compound of Formula I (e.g., a unit dose comprising aneffective concentration/amount of TPA, or of a selected pharmaceuticallyacceptable salt, isomer, enantiomer, solvate, polymorph and/or prodrugof TPA) will be readily determined by those of ordinary skill in theart, depending on clinical and patient-specific factors. Suitableeffective unit dosage amounts of the active compounds for administrationto mammalian subjects, including humans, may range from 10 to 1500 μg,20 to 1000 μg, 25 to 750 μg, 50 to 500 μg, or 150 to 500 μg. In certainembodiments, the cytopathic disease treating effective dosage of aphorbol ester compound or related or derivative compound of Formula Imay be selected within narrower ranges of, for example, 10 to 25 μg,30-50 μg, 75 to 100 μg, 100 to 250 μg, or 250 to 500 μg. These and othereffective unit dosage amounts may be administered in a single dose, orin the form of multiple daily, weekly or monthly doses, for example in adosing regimen comprising from 1 to 5, or 2 to 3, doses administered perday, per week, or per month. In one exemplary embodiment, dosages of 10to 30 μg, 30 to 50 μg, 50 to 100 μg, 100 to 250 μg, or 250 to 500 μg,are administered one, two, three, four, or five times per day. In moredetailed embodiments, dosages of 50-100 μg, 100-250 μg, 250-400 μg, or400-600 μg are administered once or twice daily. In a furtherembodiment, dosages of 50-100 μg, 100-2500 μg, 250-400μg, or 400-600μgare administered every other day. In alternate embodiments, dosages arecalculated based on body weight, and may be administered, for example,in amounts from about 0.5 μg/sq·m to about 100μ/sq·m per day, 1μg/sq·mto about 75 μg/sq·m per day, 1 μg/sq·m to about 50 μg/sq·m per day, 2μg/sq·m to about 50 μg/sq·m per day, 2 μg/sq·m to about 30 μg/sq·m perday or 3 μg/sq·m to about 30 μg/sq·m per day.

The amount, timing and mode of delivery of compositions of the inventioncomprising a cytopathic disease treating effective amount of a phorbolester compound of Formula I (AIDS treating, HIV preventing, HIVtreating, HIV reservoir activating, Th1 cytokine increasing, ERKphosphorylation inducing, chemotherapeutic, anti-tumor, cancer treating,remission inducing, remission maintaining, apoptosis inducing effectiveamount) will be routinely adjusted on an individual basis, depending onsuch factors as weight, age, gender, and condition of the individual,the acuteness of the cytopathic disease and/or related symptoms, whetherthe administration is prophylactic or therapeutic, and on the basis ofother factors known to effect drug delivery, absorption,pharmacokinetics, including half-life, and efficacy.

An effective dose or multi-dose treatment regimen for the instantcytopathic disease treating (alternatively, “AIDS treating”, “HIVtreating”, “HIV preventing”, “HIV reservoir activating”, or “Th1cytokine increasing”, “ERK phosphorylation inducing”,“chemotherapeutic”, “anti-tumor”, “cancer treating”, “apoptosisinducing”, “remission inducing”, “remission maintaining”) formulationsof the invention will ordinarily be selected to approximate a minimaldosing regimen that is necessary and sufficient to substantially preventor alleviate the symptoms of the cytopathic disease including AIDS orneoplastic diseases such as cancer and related opportunistic diseases inthe subject, and/or substantially prevent or alleviate one or moresymptoms associated with AIDS or neoplastic diseases such as cancer inthe subject. A dosage and administration protocol will often includerepeated dosing therapy over a course of several days or even one ormore weeks or years. An effective treatment regime may also involveprophylactic dosage administered on a day or multi-dose per day basislasting over the course of days, weeks, months or even years.

Various assays and models systems can be readily employed to determinethe therapeutic effectiveness of the treatment of cytopathic diseases.For example in the treatment of HIV or AIDS effectiveness may bedemonstrated by a decrease in viral load, an increase in CD4 counts, anincrease in CD3 counts, an increase in IL-2 and IFN production, adecrease in IL-4 and IL-10 production, and a decrease or elimination ofthe symptoms of AIDS among other methods of determining effectivenessknown to those of skill in the art.

Effectiveness of the compositions and methods of the invention may bedemonstrated, for example, through blood tests for HIV antibodies, viralload, CD4 levels, CD8 counts, and CD3 counts. Normal levels of CD4 areusually between 600 and 1200 per microliter, or 32-68% of lymphocytes.Individuals with a CD4 count of less than 350 have a weakened immunesystem. Those with a CD4 count of less than 200 are considered to haveAIDS. CD8 levels in a healthy individual are generally between 150-1000per microliter. CD3 levels in a healthy individual are generally betweenabout 885-2270 per microliter. Levels of CD3, CD4 and CD8 cells may bemeasured, for example, using flow cytometry. Effective amounts of thecompositions of the invention will increase levels of CD3, CD4 and CD8positive cells by at least 10%, 20%, 30%, 50% or greater reduction, upto a 75-90%, or 95% or greater. Effective amounts will also move theCD3, CD4 and CD8profile of an individual towards the optimal categoryfor each type of glycoprotein.

Individuals may also be evaluated using a beta₂-microglobulin (beta₂-M)test. Beta₂-microglobulin is a protein released into the blood when acell dies. A rising beta₂-M blood level can be used to measure theprogression of AIDS. Effective amounts of a composition of the presentinvention will lead to a decrease or cessation of increase in the amountof beta₂-M.

Effectiveness may further be demonstrated using a complete blood count(CBC). The measurements taken in a CBC include a white blood cell count(WBC), a red blood cell count (RBC), the red cell distribution width,the hematocrit, and the amount of hemoglobin. Specific AIDS-relatedsigns in a CBC include a low hematocrit, a sharp decrease in the numberof blood platelets, and a low level of neutrophils. An effective amountof a composition of the present invention will increase the levelsmeasured in a complete blood count by 10%, 20%, 30%, 50% or greaterincrease, up to 75-90%, or 95% or greater. Effective amounts will alsomove the blood protein of an individual towards the optimal category foreach type of protein.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms of HIV or AIDS including,but not limited to, oral lesions, fatigue, skin thrush, fever, lack ofappetite, diarrhea, apthous ulcers, malabsorption, thrombocytopenia,weight loss, anemia, and lymph node enlargement.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the susceptibility to and severity ofsecondary or opportunistic conditions such as mycobacterium aviumcomplex, salmonellosis, syphilis, neuroshyphilis, turberculosis (TB),bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis,listeriosis, pelvic inflammatory disease, Burkitt's lymphoma,cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma, lymphoma,systemic non-Hodgkin's lymphoma (NHL), primary CNS lymphoma,cryptosporidiosis, isosporiasis, microsporidiosis, pneumocystis cariniipneumonia (PCP), toxoplasmosis, cytomegalovirus (CMV), hepatitis, herpessimplex, herpes zoster, human papiloma virus (HPV, genital warts,cervical cancer), molluscum contagiosum, oral hairy leukoplakia (OHL),and progressive multifocal leukoencephalopathy (PML).

Effectiveness may further be demonstrated by reduction of detectable HIVin the HIV-infected subject; maintaining a normal T cell count; ormaintaining normal p24 antigen levels.

Effectiveness in the treatment of neoplastic diseases may also bedetermined by a number of methods such as, but not limited to, ECOGPerformance Scale, the Karnofsky Performance Scale, microscopicexamination of blood cells, bone marrow aspiration and biopsy,cytogenetic analysis, biopsy, immunophenotyping, blood chemistrystudies, a complete blood count, lymph node biopsy, peripheral bloodsmear, visual analysis of a tumor or lesion, or any other method ofevaluating and/or diagnosing malignancies and tumor progression known tothose of skill in the art.

For example, effectiveness of the compositions and methods herein in thetreatment of hematologic malignancies/bone marrow disorders may beevaluated using, an absolute neutrophil count (ANC). A normal ANC isbetween 1,500 to 8,000/mm3.Individuals suffering from hematologicmalignancies/bone marrow disorders frequently have an ANC below1500/mm³, and may even reach levels below 500/mm³. Effective amounts ofthe compositions and methods herein will increase an individual's ANC by10%, 20%, 30%, 50% or greater increase, up to a 75-90%, or 95% orgreater. Effective amounts may increase ANC levels above 1500/mm³.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may further be evaluatedusing, for example, a platelet count. A platelet count is normallybetween 150,000 to 450,000 platelets per microliter (×10-6/Liter).Individuals suffering from hematologic malignancies/bone marrow disordermay have platelet counts below 100,000 per microliter. Effective amountsof the compositions and methods herein will increase an individual'splatelet count by 10%, 20%, 30%, 50% or greater increase, up to a75-90%, or 95% or greater. Effective amounts may increase plateletlevels above 100,000 per microliter.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may additionally beevaluated, for example, by measuring the number of myeloblasts.Myeloblasts normally represent less than 5% of the cells in the bonemarrow but should not be present in circulating blood. Effective amountsof the compositions and methods herein will decrease the number ofmyeloblasts by 10%, 20%, 30%, 50% or more, up to a 75-90%, or 96% orgreater decrease. Effective amounts may decrease myeloblasts to below5%.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may further be evaluatedby examining myeloblasts for the presence of Auer rods. Effectiveamounts of the compositions of the present invention will decrease thenumber of Auer rods visible by 10%, 20%, 30%, 50% or more, up to a75-90%, or 96% or greater decrease up to the complete elimination ofAuer rods.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms of subjects suffering fromneoplastic disease including, but not limited to, anemia; chronicfatigue; excessive or easy bleeding, such as bleeding of the nose, gums,and under the skin; easy bruising, particularly bruising with noapparent cause; shortness of breath; petechiae; recurrent fever; swollengums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and weakness.

For each of the indicated conditions described herein, test subjectswill exhibit a 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%,or 96% or greater, reduction, in one or more symptom(s) caused by, orassociated with, the cytopathic disease, or related diseases orconditions in the subject, compared to placebo-treated or other suitablecontrol subjects.

Within additional aspects of the invention, combinatorial cytopathicdisease treating (AIDS treating, HIV preventing, HIV treating, HIVreservoir activating, Th1 cytokine increasing, ERK phosphorylationinducing, apoptosis inducing, chemotherapeutic, anti-tumor, cancertreating, remission inducing, remission maintaining) formulations andcoordinate administration methods are provided which employ an effectiveamount of a phorbol ester compound of Formula I and one or moresecondary or adjunctive agent(s) that is/are combinatorially formulatedor coordinately administered with the phorbol ester compound of FormulaI to yield a combined, multi-active cytopathic disease treatingcomposition or coordinate treatment method.

Exemplary combinatorial formulations and coordinate treatment methods inthis context employ the phorbol ester of Formula I in combination withthe one or more secondary anti-AIDS agent(s), or with one or moreadjunctive therapeutic agent(s) that is/are useful for treatment orprophylaxis of the targeted (or associated) disease, condition and/orsymptom(s) in the selected combinatorial formulation or coordinatetreatment regimen. For most combinatorial formulations and coordinatetreatment methods of the invention, a phorbol ester compound of FormulaI or related or derivative compound is formulated, or coordinatelyadministered, in combination with one or more secondary or adjunctivetherapeutic agent(s), to yield a combined formulation or coordinatetreatment method that is combinatorially effective or coordinatelyuseful to treat HIV/AIDS and/or one or more symptom(s) of aopportunistic or secondary disease or condition in the subject.Exemplary combinatorial formulations and coordinate treatment methods inthis context employ a phorbol ester compound of Formula I in combinationwith one or more secondary or adjunctive therapeutic agents selectedfrom, e.g., protease inhibitors, including, but not limited to,saquinavir, indinavir, ritonavir, nelfinavir, atazanavir, darunavir,fosamprenavir, tipranavir and amprenavir; nucleoside reversetranscriptase inhibitors including but not limited to, zidovudine,didanosine, stavudine, lamivudine, zalcitabine, emtricitabine, tenofovirdisoproxil fumarate, AVX754 and abacavir; non-nucleoside reversetranscriptase inhibitors including, but not limited to, nevaripine,delavirdine, calanolide A, TMC125 and efavirenz; combination drugsincluding, but not limited to, efavirenz/emtricitabine/tenofovirdisoproxil fumarate, lamivudine/zidovudine, abacavir/lamivudine,abacavir/lamivudine/zidovudine, emtricitabine/tenoforvir disoproxilfumarate, sulfamethoxazole/trimethoprim, and lopinavir/ritonavir; entryand fusion inhibitors, including, but not limited to, enfuvirtide,AMD070BMS-488043 fozivudine tidoxil, GSK-873,140,PRO 140,PRO 542,PeptideT, SCH-D, TNX-355, and UK-427, 857; treatments for opportunisticinfections and other conditions associated with AIDS and HIV including,but not limited to, acyclovir, adefovir dipivoxil, aldesleukin,amphotericin b, azithromycin, calcium hydroxylapatite, clarithromycin,doxorubicin, dronabinol, entecavir, epoetin alfa, etoposide,fluconazole, ganciclovir, immunoglobulins, interferon alfa-2isoniazid,itraconazole, megestrol, paclitaxel, peginterferon alfa-2pentamidine,poly-1-lactic acid, ribavirin, rifabutin, rifampin, somatropin,testosterone, trimetrexate, and valganciclovir; integrase inhibitorsincluding, but not limited to, GS 9137, MK-0518; microbicides,including, but not limited to, BMS-378806, C31G, carbopol 974P,carrageenan, cellulose sulfate, cyanovirin-N, dextran sulfate,hydroxyethyl cellulose, PRO 2000, SPL7013, tenofovir, UC-781 and IL-2.

Additional exemplary combinatorial formulations and coordinate treatmentmethods may additionally employ the phorbol ester of Formula I incombination with one or more secondary anti-tumor agent(s), or with oneor more adjunctive therapeutic agent(s) that is/are useful for treatmentor prophylaxis of the targeted (or associated) disease, condition and/orsymptom(s) in the selected combinatorial formulation or coordinatetreatment regimen. For most combinatorial formulations and coordinatetreatment methods of the invention, a phorbol ester compound of FormulaI or related or derivative compound is formulated, or coordinatelyadministered, in combination with one or more secondary or adjunctivetherapeutic agent(s), to yield a combined formulation or coordinatetreatment method that is combinatorially effective or coordinatelyuseful to treat neoplastic diseases and one or more symptom(s) of asecondary disease or condition in the subject. Exemplary combinatorialformulations and coordinate treatment methods in this context employ aphorbol ester compound of Formula I in combination with one or moresecondary or adjunctive therapeutic agents selected from, e.g.,chemotherapeutic agents, anti-inflammatory agents, doxorubicin, vitaminD3, cytarabine, cytosine arabinoside, daunorubicin, cyclophosphamide,gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone,thioguanine, aldesleukin, asparaginase, carboplatin, etoposidephosphate, fludarabine, methotrexate, etoposide, dexamethasone, andcholine magnesium trisalicylate. In addition, adjunctive or secondarytherapies may be used such as, but not limited to, radiation treatment,hormone therapy and surgery.

In certain embodiments the invention provides combinatorial cytopathicdisease treating (AIDS treating, HIV preventing, HIV treating, HIVreservoir activating, Th1 cytokine increasing, ERK phosphorylationinducing, apoptosis inducing, chemotherapeutic, anti-tumor, cancertreating, remission inducing, remission maintaining) formulationscomprising a phorbol ester and one or more adjunctive agent(s) havingcytopathic disease treating activity. Within such combinatorialformulations, a phorbol ester of Formula I and the adjunctive agent(s)having cytopathic disease treating activity will be present in acombined formulation in cytopathic disease treating (AIDS treating, HIVpreventing, HIV treating, HIV reservoir activating, Th1 cytokineincreasing, apoptosis inducing, ERK phosphorylation inducing,chemotherapeutic, anti-tumor, cancer treating, remission inducing,remission maintaining) effective amounts, alone or in combination. Inexemplary embodiments, a phorbol ester compound of Formula I and anon-phorbol ester agent(s) will each be present in a cytopathic diseasetreating amount (i.e., in singular dosage which will alone elicit adetectable alleviation of symptoms in the subject). Alternatively, thecombinatorial formulation may comprise one or both the phorbol estercompound of Formula I and the non-phorbol ester agents insub-therapeutic singular dosage amount(s), wherein the combinatorialformulation comprising both agents features a combined dosage of bothagents that is collectively effective in eliciting a cytopathic diseaseor condition symptom alleviating response. Thus, one or both of thephorbol ester of Formula I and non-phorbol ester agents may be presentin the formulation, or administered in a coordinate administrationprotocol, at a sub-therapeutic dose, but collectively in the formulationor method they elicit a detectable decrease in symptoms of cytopathicdisease in the subject. For example, in some embodiments, thecombinatorial formulation may include one or more compounds from ahighly active antiretroviral therapy protocol (HAART protocols) incombination with a phorbol ester, among other combinations. Othercombinatorial formulations may, for example, include a phorbol esterand/or compounds effective in treating the opportunistic infections ofAIDS as well as compounds from HAART protocols. In other embodiments,the combinatorial formulation may include one or more additionalchemotherapeutic agents.

To practice coordinate administration methods of the invention, aphorbol ester compound of Formula I may be administered, simultaneouslyor sequentially, in a coordinate treatment protocol with one or more ofthe secondary or adjunctive therapeutic agents contemplated herein.Thus, in certain embodiments a compound is administered coordinatelywith a non-phorbol ester agent, or any other secondary or adjunctivetherapeutic agent contemplated herein, using separate formulations or acombinatorial formulation as described above (i.e., comprising both aphorbol ester compound of Formula I or related or derivative compound,and a non-phorbol ester therapeutic agent). This coordinateadministration may be done simultaneously or sequentially in eitherorder, and there may be a time period while only one or both (or all)active therapeutic agents individually and/or collectively exert theirbiological activities.

In one embodiment, such coordinate treatment methods may, for example,follow or be derived from various highly active antiretroviral therapyprotocols (HAART protocols) and include regimens such as, but notlimited to, two nucleoside analogue reverse transcriptase inhibitorsplus one or more protease inhibitor or non-nucleoside analogue reversetranscriptase inhibitor with a phorbol ester of Formula I, among othercombinations. Other coordinate treatment methods may, for example,include a phorbol ester and/or treatments for opportunistic infectionsas well as compounds from HAART protocols. A distinguishing aspect ofall such coordinate treatment methods is that the phorbol ester compoundof Formula I exerts at least some activity, which yields a favorableclinical response in conjunction with a complementary AIDS symptomdecreasing, or distinct, clinical response provided by the secondary oradjunctive therapeutic agent. Often, the coordinate administration ofthe phorbol ester compound of Formula I with the secondary or adjunctivetherapeutic agent will yield improved therapeutic or prophylacticresults in the subject beyond a therapeutic effect elicited by thephorbol ester compound of Formula I, or the secondary or adjunctivetherapeutic agent administered alone. This qualification contemplatesboth direct effects, as well as indirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I willbe coordinately administered (simultaneously or sequentially, incombined or separate formulation(s)), with one or more secondary HIVtreating agents, or other indicated or adjunctive therapeutic agents,e.g., selected from, for example, protease inhibitors, including, butnot limited to, saquinavir, indinavir, ritonavir, nelfinavir,atazanavir, darunavir, fosamprenavir, tipranavir and amprenavir;nucleoside reverse transcriptase inhibitors including but not limitedto, zidovudine, didanosine, stavudine, lamivudine, zalcitabine,emtricitabine, tenofovir disoproxil fumarate, AVX754 and abacavir;non-nucleoside reverse transcriptase inhibitors including, but notlimited to, nevaripine, delavirdine, calanolide A, TMC125 and efavirenz;combination drugs including, but not limited to,efavirenz/emtricitabine/tenofovir disoproxil fumarate,lamivudine/zidovudine, abacavir/lamivudine,abacavir/lamivudine/zidovudine, emtricitabine/tenoforvir disoproxilfumarate, sulfamethoxazole/trimethoprim, and lopinavir/ritonavir; entryand fusion inhibitors, including, but not limited to, enfuvirtide,AMD070, BMS-488043, fozivudine tidoxil, GSK-873,140, PRO 140, PRO 542,Peptide T, SCH-D, TNX-355, and UK-427, 857; treatments for opportunisticinfections and other conditions associated with AIDS and HIV including,but not limited to, acyclovir, adefovir dipivoxil, aldesleukin,amphotericin b, azithromycin, calcium hydroxylapatite, clarithromycin,doxorubicin, dronabinol, entecavir, epoetin alfa, etoposide,fluconazole, ganciclovir, immunoglobulins, interferon alfa-2, isoniazid,itraconazole, megestrol, paclitaxel, peginterferon alfa-2, pentamidine,poly-1-lactic acid, ribavirin, rifabutin, rifampin, somatropin,testosterone, trimetrexate, and valganciclovir; integrase inhibitorsincluding, but not limited to, GS 9137, MK-0518; microbicides,including, but not limited to, BMS-378806, C31G, carbopol 974P,carrageenan, cellulose sulfate, cyanovirin-N, dextran sulfate,hydroxyethyl cellulose, PRO 2000SPL7013, tenofovir, UC-781 and IL-2.

In another embodiment, such coordinate treatment methods may, forexample, follow or be derived from various chemotherapeutic protocols.Other coordinate treatment methods may, for example, include a phorbolester and/or treatments for additional symptoms of neoplastic diseases.A distinguishing aspect of all such coordinate treatment methods is thatthe phorbol ester compound of Formula I exerts at least some activity,which yields a favorable clinical response in conjunction with acomplementary neoplastic disease symptom decreasing, or distinct,clinical response provided by the secondary or adjunctive therapeuticagent. Often, the coordinate administration of the phorbol estercompound of Formula I with the secondary or adjunctive therapeutic agentwill yield improved therapeutic or prophylactic results in the subjectbeyond a therapeutic effect elicited by the phorbol ester compound ofFormula I, or the secondary or adjunctive therapeutic agent administeredalone. This qualification contemplates both direct effects as well asindirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I willbe coordinately administered (simultaneously or sequentially, incombined or separate formulation(s)), with one or more secondary cancertreating agents, or other indicated or adjunctive therapeutic agents,e.g. doxorubicin, vitamin D3, cytarabine, cytosine arabinoside,daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin,mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase,carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide,dexamethasone, and choline magnesium trisalicylate.

As noted above, in all of the various embodiments of the inventioncontemplated herein, the cytopathic disease treating methods andformulations may employ a phorbol ester compound of Formula I in any ofa variety of forms, including any one or combination of the subjectcompound's pharmaceutically acceptable salts, solvates, isomers,enantiomers, polymorphs, solvates, hydrates, and/or prodrugs. Inexemplary embodiments of the invention, TPA is employed within thetherapeutic formulations and methods for illustrative purposes.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended therapeutic orprophylactic purpose. Suitable routes of administration for thecompositions of the invention include, but are not limited to,conventional delivery routes, devices and methods including injectablemethods such as, but not limited to, intravenous, intramuscular,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intraarterial, subcutaneous and intranasal routes.

The compositions of the present invention may further include apharmaceutically acceptable carrier appropriate for the particular modeof administration being employed. Dosage forms of the compositions ofthe present invention include excipients recognized in the art ofpharmaceutical compounding as being suitable for the preparation ofdosage units as discussed above. Such excipients include, withoutintended limitation, binders, fillers, lubricants, emulsifiers,suspending agents, sweeteners, flavorings, preservatives, buffers,wetting agents, disintegrants, effervescent agents and otherconventional excipients and additives.

If desired, the compositions of the invention can be administered in acontrolled release form by use of a slow release carrier, such as ahydrophilic, slow release polymer. Exemplary controlled release agentsin this context include, but are not limited to, hydroxypropyl methylcellulose, having a viscosity in the range of about 100 cps to about100,000 cps or other biocompatible matrices such as cholesterol.

Some phorbol ester compositions of Formula I of the invention aredesigned for parenteral administration, e.g. to be administeredintravenously, intramuscularly, subcutaneously or intraperitoneally,including aqueous and non-aqueous sterile injectable solutions which,like many other contemplated compositions of the invention, mayoptionally contain anti-oxidants, buffers, bacteriostats and/or soluteswhich render the formulation isotonic with the blood of the mammaliansubject; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and/or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers. Additionalcompositions and formulations of the invention may include polymers forextended release following parenteral administration. The parenteralpreparations may be solutions, dispersions or emulsions suitable forsuch administration. The subject agents may also be formulated intopolymers for extended release following parenteral administration.Pharmaceutically acceptable formulations and ingredients will typicallybe sterile or readily sterilizable, biologically inert, and easilyadministered. Such polymeric materials are well known to those ofordinary skill in the pharmaceutical compounding arts. Parenteralpreparations typically contain buffering agents and preservatives, andinjectable fluids that are pharmaceutically and physiologicallyacceptable such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like. Extemporaneous injectionsolutions, emulsions and suspensions may be prepared from sterilepowders, granules and tablets of the kind previously described.Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as described herein above, or an appropriatefraction thereof, of the active ingredient(s).

In more detailed embodiments, compositions of the invention may comprisea phorbol ester compound of Formula I encapsulated for delivery inmicrocapsules, microparticles, or microspheres, prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively; in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules); or withinmacroemulsions.

As noted above, in certain embodiments the methods and compositions ofthe invention may employ pharmaceutically acceptable salts, e.g., acidaddition or base salts of the above-described phorbol ester compounds ofFormula I and/or related or derivative compounds. Examples ofpharmaceutically acceptable addition salts include inorganic and organicacid addition salts. Suitable acid addition salts are formed from acidswhich form non-toxic salts, for example, hydrochloride, hydrobromide,hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, andhydrogen phosphate salts. Additional pharmaceutically acceptable saltsinclude, but are not limited to, metal salts such as sodium salts,potassium salts, cesium salts and the like; alkaline earth metals suchas calcium salts, magnesium salts and the like; organic amine salts suchas triethylamine salts, pyridine salts, picoline salts, ethanolaminesalts, triethanolamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts and the like; organic acid salts suchas acetate, citrate, lactate, succinate, tartrate, maleate, fumarate,mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, andformate salts; sulfonates such as methanesulfonate, benzenesulfonate,and p-toluenesulfonate salts; and amino acid salts such as arginate,asparginate, glutamate, tartrate, and gluconate salts. Suitable basesalts are formed from bases that form non-toxic salts, for examplealuminum, calcium, lithium, magnesium, potassium, sodium, zinc anddiethanolamine salts.

Other detailed embodiments, the methods and compositions of theinvention for employ prodrugs of phorbol esters of Formula I. Prodrugsare considered to be any covalently bonded carriers which release theactive parent drug in vivo. Examples of prodrugs useful within theinvention include esters or amides with hydroxyalkyl or aminoalkyl as asubstituent, and these may be prepared by reacting such compounds asdescribed above with anhydrides such as succinic anhydride.

The invention disclosed herein will also be understood to encompassmethods and compositions comprising phorbol esters of Formula I using invivo metabolic products of the said compounds (either generated in vivoafter administration of the subject precursor compound, or directlyadministered in the form of the metabolic product itself). Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification and the like of the administered compound,primarily due to enzymatic processes. Accordingly, the inventionincludes methods and compositions of the invention employing compoundsproduced by a process comprising contacting a phorbol ester compound ofFormula I with a mammalian subject for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radiolabelled compound of the invention,administering it parenterally in a detectable dose to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur and isolating its conversion products from theurine, blood or other biological samples.

The invention disclosed herein will also be understood to encompassdiagnostic compositions for diagnosing the risk level, presence,severity, or treatment indicia of, or otherwise managing cytopathicdiseases including, but not limited to, neoplastic diseases includingmalignant neoplastic diseases such as leukemia, and an AIDS or a relateddisease or condition in a mammalian subject, comprising contacting alabeled (e.g., isotopically labeled, fluorescent labeled or otherlabeled to permit detection of the labeled compound using conventionalmethods) phorbol ester compound of Formula I to a mammalian subject(e.g., to a cell, tissue, organ, or individual) at risk or presentingwith one or more symptom(s) or cancer and/or AIDS, and thereafterdetecting the presence, location, metabolism, and/or binding state(e.g., detecting binding to an unlabeled binding partner involved in HIVreceptor physiology/metabolism or malignant cell receptorphysiology/metabolism) of the labeled compound using any of a broadarray of known assays and labeling/detection methods. In exemplaryembodiments, a phorbol ester compound of Formula I isisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl,respectively. The isotopically-labeled compound is then administered toan individual or other subject and subsequently detected as describedabove, yielding useful diagnostic and/or therapeutic management data,according to conventional techniques.

EXAMPLES

The experiments described below demonstrate novel and powerful uses forphorbol esters and derivative compounds as HIV treating drugs that caneffectively decrease the symptoms of AIDS. In exemplary clinical trials,individuals who were unresponsive to traditional treatments for HIV andAIDS were responsive to treatments with TPA. The treatment with TPA wasallowed as “compassionate” and recovery of some patients was consideredlife-saving according to the attending physicians. The experimentsdescribed below additionally demonstrate the usefulness of phorbolesters and derivative compounds in the treatment of neoplastic diseases.These and additional findings are further expanded and elucidated withinthe following examples.

Example I Effect of TPA on the Peripheral White Blood Cells (WBC) andHemoglobin (Hb) Counts in S180 Cell-Injected Mice:

Sarcoma 180 (S180) cells were injected into Kwen-Ming mice. On the thirdday, the mice were given TPA interperitoneally (i.p.) at 50, 100 or 200μg/kg/day for 7 days. On the second day after the treatment wascompleted, blood samples were taken from the tails of the treated micefor WBC and Hb analyses. The WBC counts for the treated groups (50, 100,or 200 ug/kg/day for 7 days) were 16.1±7.4, 18.7±3.0 and 20.7±3.4×10⁹/L,respectively; the WBC count for the control group was 13.6±1.8×10⁹/L.The Hb of the treated groups were 136±11, 149±12 and 149±10 g/L, and theHb of the control group was 134+−15 g/L. The results indicate that i.p.injection of TPA could increase the peripheral WBC counts in mice in adose-dependent manner, whereas the Hb levels were not greatly affectedin TPA treated mice when compared to the control mice.

Example II Dose Ranging Study

Due to the strong local irritation caused by TPA application, TPA wasgiven to patients by intravenous (i.v.) infusion. TPA solution in asterile syringe was injected into 200 ml of sterile saline and mixedwell of i.v. infusion.

The Toxicity and Side Effects of Different TPA Doses AdministeredClinically:

(1) TPA Given at 1 mg/Patient/Week:

One mg TPA in solution was mixed well with 200 ml of sterile saline forintravenous infusion which was completed in 1 h at the rate of 16μg/min. One hour after TPA administration, patients started to havechills which lasted for about 30 min, followed by fever, (the patients'temperature reached 37.5-39.5° C. which lasted for 3-5 h, then returnedto normal) with light to heavy perspiration. The above symptoms could bealleviated by giving the patients glucocorticoids. TPA at this dosecaused a minority of patients to bleed, several patients suffered for ashort period of time difficulty in breathing, and Hb was detected in theurine. However, these side effects were short lived and reversible. Thecardiac, hepatic, renal and pulmonary functions were all found to benormal.

(2) TPA Given at 0.5 mg/Patient×2/Week: (Two Doses a Week)

0.5 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 8 μg/min.The reactions after administration were similar to that of the 1 mg TPAdosage, but to a lesser extent than the 1 mg dose. The patientstolerated the lower dose more easily. Occasionally, Hb was detected inpatients' urine. Difficulty in breathing was not observed. The cardiac,hepatic, renal and pulmonary functions were all normal.

(3) TPA Given at 0.25 mg/Patient×4/Week:

0.25 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 4 μg/min.After administration, symptoms such as chills and fever were alsoobserved, but to a much lesser extent than with the higher dosages. NoHb was detected in the urine, and no patient suffered difficulty inbreathing. The cardiac, hepatic, renal and pulmonary functions were allnormal.

Example III First Clinical Study of HIV+ patients treated with TPA

Twelve symptomatic patients (five males and seven females) between theages of 35 to 52 all of whom were infected with HIV in 1995 throughblood transfusion and were refractory to standard treatments for HIVwere treated with TPA. Each patient was administered a weight adjusteddosage of TPA (75 μg/sq m) in 200 ml of sterile saline by i.v. over onehour. This dose was administered once daily for the first three days oftreatment. Each patient was then given this dose every other day fordays 4 to 18 followed by a six month rest period prior to a secondcourse of treatment according to the same protocol.

Blood samples were gathered prior to administration of the first dose ofTPA and on days 4 and 40 of the treatment cycle. Levels of CD3, CD4 andCD8 in peripheral blood were measured using monoclonal antibodies(Becton Dickson Scientific Co., Franklin Lakes, N.J.) and a flowcytometer (B.D. Bioscience, San Diego, Calif.).

As can be seen in Table 1, no consistent change or correlation wasobserved in CD3, CD4, or CD8 levels.

TABLE ONE CD₄ CD₈ CD₃ TEST RESULTS OF TWELVE HIV PATIENTS PA- TIENT NOTEST TIME CD₄ CD₈ CD₃ 01-1 Before TPA 3 196 341 01-2 Four days after TPA3 180 299 01-3 Forty two days after TPA 2 111 203 02-1 Before TPA 26 614687 02-2 Four days after TPA 105 <2000 2616 02-3 Forty two days afterTPA 54 700 799 03-1 Before TPA 32 524 543 03-2 Four days after TPA 36366 427 03-3 Forty two days after TPA 33 374 424 04-1 Before TPA 173 735975 04-2 Four days after TPA 123 770 941 04-3 Forty two days after TPA44 493 581 05-1 Before TPA 106 1556 1646 05-2 Four days after TPA 1191330 1282 05-3 Forty two days after TPA 191 1429 1643 06-1 Before TPA232 865 1221 06-2 Four days after TPA 179 570 808 06-3 Forty two daysafter TPA 49 429 537 07-1 Before TPA 10 988 1022 07-2 Four days afterTPA 7 570 598 07-3 Forty two days after TPA 1 139 146 08-1 Before TPA524 725 1332 08-2 Four days after TPA 318 355 739 08-3 Forty two daysafter TPA 241 527 868 09-1 Before TPA 442 1021 1479 09-2 After TPA 663<2000 2920 10-1 Before TPA 407 328 778 10-2 After TPA 445 591 1077 11-1Before TPA 40 322 373 11-2 After TPA 131 724 874 12-1 Before TPA 84 256375 12-2 After TPA 78 268 362

As can be seen in Table 2, below, there were similarly inconsistentresults in the change of viral load with five patients having anincrease in HIV and no change or a reduction in seven others.

TABLE TWO BLOOD HIV COUNT OF THE TWELVE PATIENTS BEFORE DURING AND AFTERTHE TPA TREATMENT PA- TIENT RESULTS LOG FOOT NO TEST TIME (copies/ml)VALUE NOTE 01-1 3 days before TPA 3.36 × 10⁵ 5.526 01-2 4 days afterinitial TPA 1.14 × 10⁴ 6.151 01-3 15 days after initial TPA 2.02 × 10⁴4.306 01-4 25 days after initial TPA 2.60 × 10⁴ 4.416 02-1 3 days beforeTPA 9.97 × 10⁴ 4.999 02-2 4 days after initial TPA 9.72 × 10⁶ 6.899 02-315 days after initial TPA 6.33 × 10⁶ 6.801 02-4 25 days after initialTPA 8.72 × 10⁶ 6.941 03-1 3 days before TPA 3.77 × 10⁵ 5.577 03-2 4 daysafter initial TPA 8.13 × 10⁴ 4.910 03-3 15 days after initial TPA 6.11 ×10³ 3.786 03-4 25 days after initial TPA 8.59 × 10⁵ 5.934 04-1 3 daysbefore TPA 1.11 × 10⁶ 6.045 04-2 4 days after initial TPA 1.75 × 10⁷7.243 04-3 15 days after initial TPA 1.11 × 10⁶ 6.614 04-4 25 days afterinitial TPA 1.21 × 10⁴ 4.084 05-1 3 days before TPA 2.49 × 10⁴ 6.63705-2 4 days after initial TPA 9.42 × 10⁵ 5.974 05-3 15 days afterinitial TPA 2.34 × 10⁷ 7.369 05-4 25 days after initial TPA 5.56 × 10⁶6.745 06-1 3 days before TPA 4.57 × 10⁵ 5.660 06-2 4 days after initialTPA 1.44 × 10⁴ 4.160 06-3 15 days after initial TPA 1.88 × 10⁵ 5.27406-4 25 days after initial TPA 2.28 × 10⁶ 6.357 07-1 3 days before TPA2.40 × 10⁵ 5.623 07-2 4 days after initial TPA 1.51 × 10⁵ 5.179 07-3 15days after initial TPA 9.74 × 10⁴ 4.988 07-4 25 days after initial TPA5.30 × 10³ 3.724 08-1 3 days before TPA 8.02 × 10⁵ 5.904 08-2 4 daysafter initial TPA 9.09 × 10⁵ 5.959 08-3 15 days after initial TPA 5.46 ×10⁶ 6.737 08-4 25 days after initial TPA 7.77 × 10⁶ 6.890 09-1 3 daysbefore TPA undetectable 09-2 25 days after TPA undetectable 10-1 3 daysbefore TPA 1.51 × 10⁴ 4.180 Sample taken from the 10-2 25 days after TPA2.79 × 10⁴ 4.446 second cycle treatment 11-1 3 days before TPA 1.59 ×10⁵ 5.201 Sample taken from the 11-2 25 days after TPA 1.25 × 10⁵ 5.096second cycle treatment 12-1 3 days before TPA 1.32 × 10⁴ 4.122 Sampletaken from the 12-2 25 days after TPA 6.27 × 10³ 3.798 second cycletreatment

Despite the lack of correlation with viral and CD3, CD4 and CD9 levels,eleven of the patients showed significant improvement followingtreatment. Eight patients became symptom free and five of them have beenin remission for 6 to 12 months. Three additional patients had adecrease in symptoms.

Example IV Second Clinical Study of HIV+ Patients Treated with TPA

Nine of the patients in Example III were given a second treatment ofTPA. Of these nine, seven were asymptomatic at the beginning of thesecond trial. A tenth patient (patient #2a) who was symptomatic and hadnot previously been treated with TPA was added to the study. Eachpatient was administered a weight adjusted dosage of TPA (75 μg/sq m) in200 ml of sterile saline intravenously over one hour. This dosage wasgiven to each patient once a day for ten consecutive days followed by arest period of ten days for three cycles and a total of 30 doses of TPA.Patients 5a, 6a, and 8a stopped taking anti-AIDS drugs one month priorto beginning the TPA treatment and beginning again one month after thethird cycle. Patients 1-4a, 7a, and 9a-10a continued taking anti-AIDSdrugs throughout the treatment.

Blood samples were taken three days prior to starting treatment, aftercompleting the first 10 day cycle of TPA infusion and again after thelast TPA infusion and CD3, CD4, CD8, WBC, RBC, HGB and platelets weremeasured.

As shown in Table 3there was an increase in CD3 in all patients afterthe first and third infusion with TPA with the highest value occurringafter the third cycle, with the exception of two patients (5a & 10a).There was a trend for increases in the CD8 and in CD4. These resultssuggest a strengthening of the immune systems with TPA treatment. Variedresults were obtained in the HIV count (Table 4). The HIV measurementsin some of the patients were below the limits of detection of the method(less than 200) while it increased somewhat in others. There was normalvariation in the measurement of WBC, RBC, HGB and platelets (Table 5).

TABLE THREE CD₄ CD₈ CD₃ TEST RESULTS OF 10 HIV PATIENTS PA- TIENT NOTEST TIME CD₄ CD₈ CD₃ 01-1 Before TPA 5 576 1071 01-2 After first 10-dayTPA infusion cycle 7 907 1323 01-3 After third 10-day TPA infusion cycle19 1129 2037 02a-1 Before TPA 26 307 339 02a-2 After first 10-day TPAinfusion cycle 76 335 476 02a-3 After third 10-day TPA infusion cycle137 543 625 03a-1 Before TPA 295 571 870 03a-2 After first 10-day TPAinfusion cycle 460 729 1200 03a-3 After third 10-day TPA infusion cycle1002 980 2033 04a-1 Before TPA 152 672 896 04a-2 After first 10-day TPAinfusion cycle 189 584 823 04a-3 After third 10-day TPA infusion cycle205 916 1193 05a-1 Before TPA 92 1097 1175 05a-2 After first 10-day TPAinfusion cycle 91 1507 1598 05a-3 After third 10-day TPA infusion cycle94 1127 1257 06a-1 Before TPA 230 378 669 06a-2 After first 10-day TPAinfusion cycle 285 429 758 06a-3 After third 10-day TPA infusion cycle276 466 938 07a-1 Before TPA 567 1736 2258 07a-2 After first 10-day TPAinfusion cycle 729 >2000 3148 07a-3 After third 10-day TPA infusioncycle 786 >2000 3347 08a-1 Before TPA 361 569 1023 08a-2 After first10-day TPA infusion cycle 519 547 1143 08a-3 After third 10-day TPAinfusion cycle 495 733 1295 09a-1 Before TPA 101 533 672 09a-2 Afterfirst 10-day TPA infusion cycle 136 574 712 09a-3 After third 10-day TPAinfusion cycle 100 1221 1317 10a-1 Before TPA 49 178 240 10a-2 Afterfirst 10-day TPA infusion cycle 74 261 333 10a-3 After third 10-day TPAinfusion cycle 63 208 308

TABLE FOUR BLOOD HIV COUNT OF THE TEN PATIENTS BEFORE DURING AND AFTERTHE THREE TENY-DAY TPA INFUSION PATIENT RESULTS LOG NO TEST TIME(copies/ml) VALUE 01-1 3 days before TPA 4.57 × 10⁶ 6.660 01-2 afterfirst cycle TPA infusion 2.99 × 10⁵ 5.475 01-3 after third cycle TPAinfusion 9.41 × 10⁵ 5.973 02a-1 3 days before TPA 2.71 × 10⁵ 5.433 02a-2after first cycle TPA infusion 3.09 × 10⁵ 5.490 02a-3 after third cycleTPA infusion 9.24 × 10⁵ 5.966 03a-1 3 days before TPA undetectable —03a-2 after first cycle TPA infusion lower the 500 2.371 03a-3 afterthird cycle TPA infusion 9.55 × 10³ 3.980 04a-1 3 days before TPA lowerthan 500 2.312 04a-2 after first cycle TPA infusion undetectable — 04a-3after third cycle TPA infusion 2.38 × 10³ 3.376 05a-1 3 days before TPAundetectable — 05a-2 after first cycle TPA infusion undetectable — 05a-3after third cycle TPA infusion undetectable — 06a-1 3 days before TPAundetectable — 06a-2 after first cycle TPA infusion undetectable — 06a-3after third cycle TPA infusion undetectable — 07a-1 3 days before TPAundetectable — 07a-2 after first cycle TPA infusion undetectable — 07a-3after third cycle TPA infusion undetectable — 08a-1 3 days before TPA1.13 × 10⁴ 4.054 08a-2 after first cycle TPA infusion 6.68 × 10⁴ 4.82508a-3 after third cycle TPA infusion 6.20 × 10⁴ 4.792 09a-1 3 daysbefore TPA 1.38 × 10⁵ 5.139 09a-2 after first cycle TPA infusion 1.65 ×10⁵ 5.217 09a-3 after third cycle TPA infusion 2.35 × 10⁵ 5.371 10a-1 3days before TPA 7.20 × 10⁵ 5.857 10a-2 after first cycle TPA infusion2.82 × 10⁵ 5.450 10a-3 after third cycle TPA infusion 1.86 × 10⁵ 5.270

TABLE FIVE PERIPHERY BLOOD COUNT OF THE TEN PATIENTS BEFORE AND AFTERTHE TPA THREE 10-DAY TREATMENT PATIENT WBC RBC HGB PLt NO TEST TIME(×10⁹/L) (×10¹²/L) (g/L) (×10⁹/L) 01-1 Before TPA 2.3 2.55 92 199 01-2After first 10-day TPA infusing 4.4 2.61 99 325 01-3 After third 10-dayTPA infusing 6.1 2.91 102 182 02a-1 Before TPA 5.7 2.44 114 227 02a-2After first 10-day TPA infusing 3.7 2.14 88 238 02a-3 After third 10-dayTPA infusing 11.1 2.52 100 124 03a-1 Before TPA 7.8 4.04 147 309 03a-2After first 10-day TPA infusing 9.8 3.83 1.38 338 03a-3 After third10-day TPA infusing 13.6 4.54 140 549 04a-1 Before TPA 3.9 3.34 127 23204a-2 After first 10-day TPA infusing 3.6 2.92 107 306 04a-3 After third10-day TPA infusing 9.2 2.85 105 105 05a-1 Before TPA 5.1 3.54 146 24305a-2 After first 10-day TPA infusing 5.7 3.46 1.35 315 05a-3 Afterthird 10-day TPA infusing 10.1 3.61 144 130 06a-1 Before TPA 5.0 4.21171 198 06a-2 After first 10-day TPA infusing 4.2 3.48 142 256 06a-3After third 10-day TPA infusing 6.5 3.66 154 169 07a-1 Before TPA 6.63.62 102 306 07a-2 After first 10-day TPA infusing 6.0 3.76 143 25807a-3 After third 10-day TPA infusing 6.0 3.92 123 293 08a-1 Before TPA3.1 4.03 125 116 08a-2 After first 10-day TPA infusing 4.3 3.86 128 22108a-3 After third 10-day TPA infusing 6.8 4.19 128 138 09a-1 Before TPA3.5 1.43 41 114 09a-2 After first 10-day TPA infusing 2.6 1.99 57 21409a-3 After third 10-day TPA infusing 4.0 2.33 67 170 10a-1 Before TPA2.6 2.65 78 297 10a-2 After first 10-day TPA infusing 2.9 2.58 92 18710a-3 After third 10-day TPA infusing 7.0 4.31 130 138

Of nine patients previously treated with TPA in the first clinicalstudy, only one (#9a) presented with some AIDS symptoms prior to thestart of the second clinical study. Following treatment with threecycles of TPA in the second study, this patient and another (#2a), whohad never been treated with TPA, experienced a disappearance of AIDSsymptoms and both became sufficiently well to resume their normalactivities. The other eight patients began the study without AIDSsymptoms and were symptom free at the end of the study. All patientsremain under observation. Treatment with anti-AIDS drugs continuesuninterrupted.

As can be seen in Table 4, there was an increase in all patients in theCD 3, 4 and 8 levels with the most striking and consistent increases inCD3 levels. The viral load of HIV varied. It was undetectable in threepatients (<200); it increased somewhat in six others and was reduced inone.

Example V Third Clinical Study of HIV+ Patients Treated with TPA

Six patients, two males and four females between the ages of 37 and 52years of age (Patients #13-18), were treated with TPA. Four of thesepatients previously received TPA treatment in combination with anti-HIVdrugs in the two previous clinical studies. The two remaining patientshad never been treated with TPA, but had previously received anti-HIVdrug regimens. All treatments were stopped three days prior to theinitiation of the third clinical study and were not resumed until 60days after completion of the TPA treatment. The resumption of thestandard HIV treatments was required by local health authorities.

Each patient in the study received 150 μg of TPA in 200 ml of sterilesaline by intravenous infusion over a 1.5 to 2 hour period daily for 60days for a total administered dose of 9.0 mg. Following completion ofthe 60 days of TPA therapy, these patients remained under observationfor an additional 60 days though they received no further treatment.

CD3, CD4 and CD8 levels in peripheral blood were quantitated prior tostarting treatment, and again at 30 and 60 days using flow cytometry andthe appropriate antibodies obtained from B.D. Bioscience, San Diego,Calif. Viral load was determined using conventional methods at Kuang Annmen Hospital, Beijing, China. Patients RBC, WBC, platelets andhemoglobin levels were also measured.

As can be seen in Table 6, the viral load in the six patients was eitherlow or undetectable at the beginning of the trial and remained lowthroughout the clinical trial period despite the discontinuation oftraditional antiretroviral therapy. Additionally, there was no reboundin viral levels 6 to15 days after stopping antiretroviral treatment aspreviously reported as occurring in patients with a plasma viral loadbelow 50 HIV copies per ml. (Harrigan et al., AIDS 13, F59-F62 (1999).The CD3, CD4 and CD8 levels were variable and inconclusive.

TABLE SIX STUDY 3 CD₄CD₈CD₃ AND HIV LOAD RESULTS OF 6 PATIENTS PATIENT*TEST **HIV # TIME CD3 CD4 CD8 (copies/ml) 13 1 3500 1135 >2000undetectable 2 2771 735 1938 0.533 3 2689 721 1897 0.133 14 1 1415 677664 0.374 2 1522 613 796 0.353 3 902 369 485 0.038 15 1 759 9 542 0.5332 1865 8 1408 1.99 3 2099 11 1507 undetectable 16 1 1368 128 1166undetectable 2 1477 105 1318 1.28 3 1305 46 1220 0.012 17 1 428 95 2970.002 2 594 112 424 0.152 3 317 31 246 0.056 18 1 1041 392 457undetectable 2 703 229 343 0.174 3 579 165 290 undetectable *Testtime: 1. Before TPA 2. Thirty days after TPA 3. Sixty days after TPA**All figures are in the million

White blood cells (WBC), red blood cells (RBC), hemoglobin (Rb) andplatelets (PLt) were measured prior to starting TPA treatment, 15, 30,45 and 60 days after starting TPA treatment and 30 days after stoppingTPA treatment. As can be seen in Table 7, most values were within thenormal range.

The patients involved in the third clinical study experienced no viralload rebound as typically seen when antiretroviral therapies arediscontinued. They additionally had no recurrence of AIDS symptomsduring the 120 day observation and treatment period, felt normal andwere able to conduct their usual life activities.

TABLE SEVEN STUDY 3 PERIPHERY BLOOD PROFILE OF 6 PATIENTS PATIENT WBCRBC Rb PLt # *TEST TIME (×10⁹/L)× (×10¹²/L) (g/L) (×10⁹/L) 13 1 9 3.75139 246 2 9 3.88 140 240 3 8.9 4.35 148 275 4 3.6 3.9 125 304 5 8.8 4.55126 221 6 7.5 4.55 130 272 14 1 4.2 4.16 111 118 2 4.1 4.03 114 169 35.9 4.48 116 232 4 3.9 4.44 109 152 5 4.4 4.31 96 227 6 6.5 4.4 104 19315 1 5.9 3.67 110 397 2 5 3.41 101 219 3 5.2 4.84 113 247 4 6.2 4.13 110262 5 6.2 4.04 99 239 6 8.4 3.9 110 278 16 1 6 3.62 144 297 2 8.1 3.65142 415 3 4.3 4.03 145 345 4 4.6 3.86 124 291 5 5.1 4.1 123 276 6 3.84.71 144 224 17 1 5.5 3.06 124 242 2 6.4 2.98 118 151 3 4 3.2 121 177 43.9 3.49 116 131 5 7.7 3.34 99 121 6 4.8 3.42 100 178 18 1 7.4 3.91 156240 2 8.1 3.69 141 208 3 4.5 4.32 154 228 4 4.9 4.14 131 149 5 3.5 4.56136 222 6 NA NA NA NA *Test time: 1. Before TPA 2. Fifteen days afterTPA 3. Thirty days after TPA 4. Forty five days after TPA 5. Sixty daysafter TPA 6. Thirty days after stop TPA

Example VI Case Studies

Results of treatment of initially symptomatic AIDS patients treated withTPA according to the protocols of Example III, IV, and V. Patients whoparticipated in multiple studies are in some cases identified by morethan one patient number. All patient identification numbers correspondto the patient numbers in Tables 1-7.

Patient #1 and 15: H. L. Y., female, 35, participated in all threeclinical studies, diagnosed with AIDS and had clear symptoms of thisdisease in 2003. At the time the first study began, she had frequentfever, diarrhea, oral lesions, poor appetite, weight loss, left eyevision loss (syncytia formation) and coughing (tuberculosis). Thepatient started to receive antiviral medications Stavudine (D₄T),Lamivudine (3TC), Nevirapine (NVP) and Zidovudine (AZT) in 2004. Despiteanti-AIDS drugs, she had a CD4 count of 3 and was unable to perform anyphysical work.

During the first study following the protocol of Example III, above, sheexperienced an increase in body temperature of 38-39° C. on fourdifferent occasions that lasted 2 to 4 hours. After treatment with TPA,there was a gradual improvement in symptoms. Her appetite improved anddiarrhea, oral lesions, and fatigue disappeared but her eyesightremained impaired. She gained some weight and reported being able toresume housework. She continues to receive antiviral therapy. Thereappears to be no correlation in improvements in symptoms and changes inher CD 3, 4, 8 level and viral count.

H. L. Y. participated in the second study described in Example IV,above. At the initiation of the second study she has no symptoms ofAIDS. During this subsequent treatment with TPA she experienced noadverse effects. After both the first and third cycle of treatment withTPA, her CD3, CD4, and CD8 levels increased as did her white blood cellcount. Her HIV count was somewhat higher, but she is able to functionnormally and continues to have no symptoms of AIDS.

H. L. Y. participated in the third study described in Example V, above.At the initiation of the third study, she was still having problems withher eye. During the third study, she experience a fever of 38-38.5° C.during the third and fourth day of TPA infusion. No AIDS symptomsreturned during either the study or the 60 day observation period.Except for her sight, she remains symptom free, feels normal and is ableto conduct normal activities. She reinitiated antiviral therapy aftercompletion of the 60 day observation period and remains under the careof a physician.

Patient #2: C. X., female, 49, participated in first clinical study,diagnosed with AIDS and had clear symptoms of this disease in 2004. Shehad mild oral lesions, fatigue, skin thrush, fever and poor appetite.Some of these symptoms were due to herpes virus. She had been treatedwith AZT, DDI and NVP but drug treatment was terminated due to sideeffects. She received no drugs for 3 months prior to TPA treatment. Shewas hospitalized frequently and was unable to work. Her CD4 count priorto treatment was 26.

During TPA treatment according to the protocol of Example III, sheexperienced an increase in body temperature of 37.5 to 38 degreescentigrade on three different occasions that lasted 1-2 hours. Aftertreatment with TPA, her oral lesions, skin thrush and fever disappeared.Her appetite improved sufficiently so that she gained weight and hadsufficient energy to resume housework. She remained symptom free forfive months and was not given any anti-AIDS drugs during this period.There appeared to be no correlation between the improvement in symptomsand her CD 3, 4, 8 levels and viral count.

Patient #2a: M. S., male, 48, participated only in the second clinicalstudy, had frequent fever, diarrhea, weight loss, a weak immune system,severe depression and was unable to work.

During treatment with TPA according to the protocols of Example IV, hisbody temperature increased to 38.5 to 39 degrees centigrade on fiveoccasions for 2 to 4 hours.

After the third cycle of TPA treatment, the fever and diarrhea were nolonger a problem. His CD3, CD4 and CD8 counts trended upwards as did theWBC and HIV count. His physical and mental condition returned to normaland he is able to work.

Patient #3: Y. P., male, 51, participated only in the first clinicalstudy, diagnosed with AIDS and had clear symptoms of this disease in2004. His major symptoms were diarrhea, fatigue, weight loss, anemia andpurple marks on the skin of both legs; and he could only do light work.He was being treated with AZT, DDI and NVP but a serious anemia resultedin the termination of drug treatment four months prior to being givenTPA. His initial CD4 count was 32.

During TPA treatment according to the protocol described in Example III,he experienced an increase in body temperature of 38 to 39° C. on threeoccasions that lasted 1 to 2 hours. After treatment with TPA, there wasa marked improvement in his symptoms and he was able to return to workinvolving heavy labor and is leading a normal life. He was symptom freefor five months after TPA therapy and was not treated with antiviraldrugs during this period. There appeared to be no correlation between CD3, 4, and 8 levels and improvement in symptoms but there was someincrease in viral count.

Patient #4: L. W., male, 34, participated in only the first clinicalstudy, tested positive for HIV and had clear symptoms of this disease in2004. His major symptoms were diarrhea, fever, weight loss, cough(tuberculosis), right side neck lymph node enlargement and he was unableto work. His initial response to treatment was poor. The schedule ofantiviral medication of 3TC, DDI and NVP was irregular and was stoppedduring TPA therapy. His initial CD4 count was 173.

During treatment with TPA according to the protocol of Example III, heexperienced an increase in body temperature of 38 to 39° C. on fiveoccasions that lasted 0.5 to 1 hours. After treatment, the occasionalbout of diarrhea was treated successfully with and an anti-diarrheadrug. An improvement in appetite has resulted in an increase in weightand energy that resulted in his returning to a regular work schedule.The lymph node returned to normal size. He continues to be treated withanti-viral drugs. There appeared to be no correlation between theimprovements in symptoms, CD3, 4, 8 levels and viral count.

Patient #5 and 3a: H. S., female, 37, participated in the first twoclinical studies, tested positive for HIV and had clear symptoms of thedisease in of 2004. At the time the first study began, her majorsymptoms were skin thrush, hair loss, mouth infection, weight loss andfatigue. She was being treated with D₄T, DDI, and NVP but treatment wasstopped due to loss of kidney function. She had an initial CD4 count of106 but could handle regular labor work.

During treatment with TPA according to the protocol of Example III, sheexperienced an increase in body temperature of 37.5 to 38° C. on fiveoccasions that lasted 0.5 to 1.0 hours. After treatment with TPA, noimprovement in symptoms occurred. Treatment with anti-viral drugs wasresumed without return of the previous side effects and the intensity ofher symptoms were reduced after one month. This treatment is beingcontinued and she has returned to work. There appeared to be nocorrelation between the improvement in symptoms and changes in the CD 3,4, and 8 levels or the HIV count.

At the time of the second study, she had no symptoms of AIDS andsuffered no adverse effects to the course of treatment described inExample IV. After the second study, her CD3, CD4 and CD8 levels trendedupwards as did her white blood count and platelet levels. Her HIV countwas initially undetectable, but increased after the third cycle oftreatment. She is currently able to work.

Patient #6, #4a, and #17: H. S. C., male, 36, participated in all threeclinical studies, tested positive for HIV and had clear but mildsymptoms in 2004. At the time the first study began, he suffered fromdizziness, headache, poor appetite and an increased susceptibility toupper respiratory tract infections but was able to work regularly as alaborer. He was being treated with antiviral drugs AZT, DDI and NVP butterminated their use due to adverse reactions. His initial CD4 level was232.

During treatment with TPA according to the protocol of Example III, hedid not experience an increase in body temperature or any other sideeffect. After treatment, his symptoms remained unchanged and a reductionin platelets appeared unrelated to TPA treatment. He continued to betreated with antiviral drugs and is able to work as before. Thereappeared to be no correlation between the improvement in symptoms andthe CD 3, 4 and 8 levels and viral load.

At the time of the second study, he had no symptoms and his immunesystem appeared to be functioning normally. During the second studyaccording to Example IV, he again suffered no side effects fromtreatment with TPA. His CD3, CD4, and CD8 count increased somewhat asdid his white blood cell count. The viral load was initiallyundetectable but increased after the third cycle of treatment. However,he does not have any symptoms of AIDS and has returned to work.

At the initiation of the third clinical study, he had no symptoms.During treatment with TPA according to the protocol of Example V, heexperienced an incident of local irritation due to a leaking needle onday 32 but was treated successfully in three days. He remains symptomfree, feels normal, and is able to do heavy labor. He started antiviraltherapy after completion of the 60 day observation periods and remainsunder the care of a physician.

Patient #7, #5a and #16: H. C. L., male, 49, participated in all threeclinical studies, tested positive for HIV and had clear symptoms of thedisease in 2004. His major symptoms at the time of the first study wereweight loss, skin thrush, fatigue, poor appetite and coughing(tuberculosis) but he was able to do light work. He was treatedsimultaneously with D₄T, DDI, NVP and antituberculosis medication. Hisinitial CD4 count was 10.

During treatment with TPA according to the protocol outlined in ExampleIII, he experienced an increase in body temperature to 38° C. on twooccasions accompanied by mild dizziness and headache. After treatment,his symptoms remained unchanged and antiviral therapy was resumed onemonth later. With time, his cough, appetite and energy level improvedand he is able to work. He continued both antiviral andanti-tuberculosis medication. There appeared to be no correlationbetween improvements in symptoms and his CD3, 4 and 8 levels or viralload.

At the time of the second clinical investigation, he had no symptoms ofAIDS and his immune system appeared to be functioning normally. Hesuffered no adverse effects from treatment TPA during the secondclinical investigation. After treatment, his CD4 level was unchanged,but his CD3 and CD8 levels trended upwards as did his white blood cellcount. His viral load was undetectable. He has not had any symptoms ofAIDS and has returned to work.

At the start of the third clinical investigation, he was notexperiencing AIDS symptoms. During treatment according to the protocoloutline in Example V, he suffered from a fever on one occasion. Heremains symptom free, feels normal, and is able to do heavy labor.

He re-started antiviral drugs after completion of the 60 day observationperiod and remains under the care of a physician.

Patient #8, #6a, and 18: Y. X. O., female, 36, participated in all threeclinical studies, tested positive for HIV in 2004. Her major symptom atthe time of the first study was an increased susceptibility to upperrespiratory tract infection. She was treated with AZT, DDI and NVP. Atthe start of the study, her CD4 level was 524 and she could handleregular labor work.

During treatment with TPA according to the protocol of Example III, sheexperienced an increase in body temperature to 38.5° C. on one occasionthat lasted four hours. After treatment, the frequency of her coldsdecreased and she had no other symptoms. She continued to be treatedwith antiviral drugs and is able to work. There appeared to be nocorrelation between the improvement in symptoms and her CD 3, 4, or 8levels or viral load.

At the time of the second clinical investigation, she had no symptoms ofAIDS and her immune system appeared to be functioning normally. Duringthe second study, according to the protocols of Example IV, her bodytemperature again rose to 38.5 degrees centigrade for two hours on asingle occasion. After treatment, her CD3 and CD8 levels increasedsomewhat while her CD4 and white blood cell count remained unchanged.Her viral load is undetectable. She appears normal and is able to workat physically demanding tasks.

At the time of the third clinical investigation she was symptom free.The only side effects from treatment according to the protocol ofExample V was as fever of 38-39° C. on the second day of the treatmentthat lasted for two hours and skin irritation from a leaking needle onday 36 that cleared in two days. She remains symptom free, feels normaland is able to do heavy labor. She re-started antiviral therapy aftercompletion of the 60 day observation period and remains under the careof a physician.

Patient #9 and #7a: C. T. F., male, 44, participated in the first twoclinical studies, tested positive for HIV and had clear symptoms of thedisease in 2004. His symptoms at the initiation of the first studyincluded persistent diarrhea, dizziness, headaches, poor appetite,weight loss and fatigue. He had a positive response to AZT, DDI and NVPtreatment and blood HIV count was near the lowest limit. Despite thepositive response, his symptoms persisted and he checked into thehospital due to diarrhea that persisted for 20 days. He was verydepressed and unable to do any work.

During treatment with TPA according to the protocol of Example III, heexperienced an increase in body temperature of 37.5 to 38° C. on sixoccasions that lasted 2 to 4 hours. A leaking needle caused a seriousskin irritation during one administration of TPA but was treatedsuccessfully. After eight treatments with TPA, the mild dizziness andheadache persisted but the incidence of diarrhea began to decrease andhis appetite improved. A week later, his diarrhea was completely goneand he had a normal appetite. He was able to return to work and isreceiving antiviral drug therapy. There appeared to be an upward trendof CD3, 4, 8 levels and the HIV count was undetectable.

At the time of the second clinical investigation, he had no symptoms ofAIDS and his immune system appeared to be functioning normally. DuringTPA treatment according to the protocol of Example IV, he suffered noadverse effects. After treatment, his CD3, CD4 and CD8 levels increasedsomewhat while his white blood cell count remained unchanged. His HIVcount continues to be undetectable. He is able to do strenuous work.

Patient #10 and #8a: W. F. W., Female, 47, participated in the first twostudies, tested positive for HIV and had clear symptoms of the diseasein 2003. Her symptoms at the initiation of the first study included lowbody temperature, diarrhea, low platelet count, coughing blood, bloodybowel movements, dizziness, headache, poor appetite, weight loss,fatigue with mild skin thrush and deep depression. She was hospitalizedon one occasion for two months because of bloody bowel movements. Shewas very depressed and unable to work. She did not respond positively tothe AZT, DDI and NVP treatment and her symptoms were not under control.

During treatment with TPA according to the protocol of Example III, sheexperienced an increase in body temperature to 38.5° C. on one occasionthat lasted 4 hours.

After TPA treatment, her dizziness, headache and diarrhea graduallylessened. Eventually, her appetite led to a weight gain and animprovement in her energy level. Her platelet count rose from 30,000 to110,000 per microliter and the skin thrush and diarrhea were eliminated.She was able to work again and was treated with antiviral drugs. She hadfever and diarrhea occasionally that she was able to control with drugs.

Six months later she suffered from mild headaches and dizziness andunderwent a second treatment with TPA. During her second treatment withTPA, she experienced an increase in body temperature to 37.5 to 38° C.on five occasions that lasted 2 to 4 hours. Twenty hours after the13^(th) injection of TPA, her temperature reached 40.5 degreescentigrade and lasted for several hours. It was concluded that theincrease in temperature was not related to TPA therapy.

After her second treatment with TPA, her symptoms disappeared, herappetite improved and she gained weight, which enabled her to regain herenergy, return to work and lead a normal life. She was free of symptomsfor one year and has had few colds in the first six months after thesecond TPA treatment. There appears to be an upward trend for the CD3,4, and 8 levels and the HIV counts.

At the time of the second clinical trial according to the protocol ofExample IV, this patient continued to display no symptoms of AIDS andher immune system appeared to be functioning normally. She suffered noadverse effects during treatment. After treatment, her

CD3, CD4 and CD8 counts increased somewhat as did her WBC. Her HIV countincreased somewhat. Since the studies, she has been healthy and engagedin laborious work.

Patient #11 and 9a: C. T. L., female, 40, participated in the first twostudies, was diagnosed with AIDS and had clear symptoms of this diseasein 2003. At the initiation of the first study she had persistentdiarrhea, low body temperature, oral lesions, severe skin thrush,itching, purple blotches on her face and lips, dizziness, headache, poorappetite, and fatigue and depression. She responded poorly to AZT, 3TCand NVP treatment. Her symptoms were not under control and she wasunable to work. Her initial CD4 count was 40.

During her first treatment with TPA, she experienced an increase in bodytemperature to 38 to 39° C. on four occasions that lasted 2 to 4 hours.She had shortness of breath on two occasions that lasted 20 to 30minutes each.

After the sixth dose of TPA, her skin thrush began to disappear and uponcompletion of TPA treatment, the dizziness, headache, fever and skinthrush were improving and gradually faded away. Her appetite, physicalcondition and depression improved sufficiently for her to return towork.

This patient had a second treatment with TPA 18 months later due to thereturn of symptoms including mild skin thrush, diarrhea and dizziness.During this second treatment, she experienced an increase in bodytemperature to 37.5 to 38° C. three times that lasted 2 to 4 hours.There were no other adverse reactions. After treatment with TPA, hersymptoms disappeared completely and her physical condition improvedsufficiently to allow her to return to work. She has been withoutsymptoms for one year and she has rarely had a cold. There appears to bean upward trend in CD3 , 4, and 8 levels, but her HIV counts did notchange.

At the time of the second clinical study according to the protocol ofExample IV, this patient exhibited symptoms of AIDS including headache,dizziness, poor appetite and a weak immune function. She suffered noadverse effects during treatment. After treatment, her CD3 and CD8levels increased while her CD4 count was unchanged. Her HIV countincreased slightly but no other changes were observed. Her mental andphysical condition has improved considerably and she is doing strenuousphysical work.

Patient #12 and #10a: C. C. L., female, 39, participated in the firsttwo studies, diagnosed with AIDS and had clear symptoms of this diseasein 2003. At the initiation of the first study she had persistent lowbody temperature, skin thrush, dizziness, headache, poor appetite, orallesions, fatigue and deep depression. She was treated with AZT, 3TC andNVP but had poor results and she was unable to work. Her initial CD4count was 84.

This patient was treated with TAP twice during the period March 2005 toMarch 2006. During the first treatment with TPA, she experienced anincrease in body temperature to 38 to 38.5° C. on eight occasions thatlasted 2 to 4 hours. She experienced shortness of breath on one occasionfor 15 minutes and suffered a skin irritation due to a leaking needle.

After the seventh injection, her oral lesions disappeared. Uponcompletion of all the injections, all symptoms disappeared and herphysical condition improved sufficiently for her to return to work.

Six months later, this patient was re-treated with TPA due to the returnof light diarrhea and dizziness. She experienced an increase in bodytemperature to 37.5 to 38° C. centigrade on six occasions associatedwith TPA administration that lasted 2 to 6 hours. Starting with theeight injection, the dose was increased from approximately 150 μg to 250μg TPA. No adverse effects occurred. Upon completion of TPA therapy, allher symptoms disappeared. Her physical condition was restored to normaland she returned to work and has had a normal life. She has been symptomfree for one year and has rarely had a cold. There were no changes in CD3, 4 or 8 levels, but her HIV count increased.

At the time of the second clinical study, this patient had no symptomsof AIDS though she did have a weakened immune system. She was treatedaccording to the protocol of Example IV and suffered no adverse effects.After treatment, there were slight increases in her CD3, CD4 and CD8,and modest increases in WBC, RBC and HGB while platelets appeared todecrease. The HIV count was reduced somewhat. She has been healthy andengaged in strenuous physical work since her treatments.

Patient #13: L. F. L., female, 53, diagnosed with AIDS in 2004,participated in only the third clinical study. She presented with mildsymptoms of poor appetite and weight loss. Long term antiviral drugswere effective and caused her virus count to decrease below detectablelevels and CD36, CD4 and CD8 counts to increase to a high level. She hadnot symptoms prior to TPA treatment and had no side effects from itsadministration. She remains symptom free, feels normal, and is able toconduct normal activities. She re-started antiviral drug therapy aftercompletion for the 60 day observation period.

Patient #14: K. S. M., female, 45, diagnosed with AIDS in 2004,participated in only the third clinical study. Her symptoms were mildand consisted of poor appetite and frequent colds. She had been treatedwith antiviral drugs, but stopped due to severe liver toxicity. She hadno symptoms prior TPA treatment and the only TPA side effect wasirritation due to a leaking needle on day 43 that was easily treated. NoAIDS symptoms occurred during the entire treatment and observationperiod. She feels normal and is able to conduct her usual activities.After completion of the 60 day observation period she was lost to thestudy and did not renew antiviral therapy.

Example VII Treatment of Relapsed/Refractory Malignancies with TPA

Patients with histologically documented relapsed/refractory hematologicmalignancy/bone marrow disorders are treated with a combination of TPA(Xichuan Pharmaceuticals, Nan Yang, Henan, China), dexamethasone andcholine magnesium trislicylate. Comparable methods as set forth belowfor demonstrating the therapeutic use of TPA in the treatment of AcuteMyelogenous Leukemia (AML) will be applied to demonstrate the use of TPAfor treating other neoplastic conditions and malignancies. Otherneoplastic conditions and malignant disorders amenable to treatmentusing the methods and compositions of the invention include variousforms of cancer, including blood and bone malignancies and solid tumorsof various types. In addition to the specific protocols herein,successful treatment and/or remission will be determined for differenttargeted neoplastic and malignant conditions using any of a wide varietyof well known cancer detection and assessment methods—for example bydetermining size reduction of solid tumors, histopathological studies toevaluate tumor growth, stage, metastatic potential, presence/expressionlevels of histological cancer markers, etc.

AML is an aggressive disease that generally warrants urgent andintensive therapy. The average patient age at AML diagnosis is 64-68years old, and patients over the age of 60 treated with standardchemotherapy are cured of their disease <20% of the time. Patients whodevelop AML after an antecedent hematologic disorder or priorleukemogenic chemotherapy/radiation therapy have similarly pooroutcomes, as do patients whose disease is associated with specificadverse cytogenetic and clinical features. Hence, most patientsdiagnosed with AML have patient and/or disease-related features that areassociated with a very poor prognosis. For patients with relapseddisease, no standard non-transplant therapy has demonstrated thecapacity for cure. For these patients, AML is often a fatal disease. Newapproaches to the therapy of AML are needed.

Employing the methods and compositions of the instant invention, TPA, isdeveloped as a therapeutic agent for treating patients with AML, basedon TPA's novel role in modulating intracellular signaling pathways, itscapacity to induce differentiation and/or apoptosis in cell lines, andclinical data indicating the effectiveness of TPA in treating neoplasticand malignant disorders, including myeloid malignancies.

Thus far clinical evaluation of TPA has demonstrated that TPA exertsdirect therapeutic cytotoxic effects in at least a subset of AML cases,as measured by cell viability and apoptosis assays. In all primarycultures analyzed by Western analysis, TPA strongly induced ERKphosphorylation by 1 hour in culture. TPA's cytotoxic effect on primaryAML cells is associated with the subsequent loss of the phosphor-ERKpro-survival signal after 24 hour ex vivo exposure. This observation isin good agreement with other studies that reported decreased primary AMLsurvival after pharmacological interruption of ERK signaling by MEKinhibitors, such as PD98059, U0126 and PD 184352. In our studies, lossof ERK signaling was associated with induction of ERK phosphatases.

In addition to protein kinase C and ERK activation, TPA is a knowninducer of NF-κB, a pro-survival transcription factor oftenconstitutively active in AML blasts and leukemic stem cells. Recent workfrom our laboratory has demonstrated that AML cell NF-κB can beinhibited in vivo with 48h of treatment with dexamethasone + cholinemagnesium trisalicylate (CMT). In addition, we have shown thatdexamethasone can induce MKP-1 ERK phosphatase expression and enhanceTPA cytotoxicity on primary AML samples. In this context, we have chosenin exemplary embodiments below to use dexamethasone and CMT asadjunctive medications to be used 24 h pre- and 24 h post treatment withTPA. These medications are well-tolerated and anticipated to reduceinflammatory adverse effects of treatment and enhance TPA cytotoxicityby increasing ERK phosphatase expression and inhibiting NF-κB. Inaddition dexamethasone and CMT will be used as adjunctive medicationsbecause they are anti-inflammatory, may ameliorate adverse effects, andmay enhance anti-leukemic activity by inhibition of the anti-apoptoticeffects of constitutive NF-κB expression and induction of phosphatasesthat decrease signaling pathway activity.

An initial TPA Phase 1 study enrolled 35 patients [23 withrelapsed/refractory AML, 2 with other myeloid malignancies (CML-blastcrisis, myelodysplasia with excess blasts), 3 with Hodgkin's Disease, 3with non-Hodgkin's lymphoma and 4 with solid tumors]. The majority ofpatients had relapsed/refractory AML. Our clinical results include oneAML patient with stable disease for ≧5 months, who received 8 TPAinfusions. In a second AML patient, a pronounced (5-fold) decline in thenumber of circulating blasts was seen following TPA administration. Thisdecline in leukemic blasts persisted for 4 weeks, and the patienteventually died from a fungal infection. Finally, a patient withrelapsed and refractory Hodgkin's disease despite high dose chemotherapywith autologous stem cell rescue had a partial remission of a chest wallmass after TPA administration. TPA dose escalation has been completed,in the last cohort 2 out of 3 patients treated at a dose of 0.188 mg/m2d1-5, 8-12 experienced grade III non-hematologic dose limitingtoxicities (DLT), establishing the maximum tolerated TPA dose as asingle agent at 0.125 mg/m2/d on d1-5 and 8-12.

In the case of AML and other hematologic malignancies, patients aregiven an initial dose of TPA of 1 mg/week×3 weeks (days 1, 8, 15)administered with continuous/intermittent pulse oximetry for 6 hours.Twenty four hours prior to initiation of TPA therapy, patients are given10 mg of dexamethasone every six hours and 1500 mg of choline magnesiumtrisalicylate (CMT) every eight hours continuing until 24 hours afteradministration of TPA. After administration of the initial dose of TPA,patients have a two week rest period after which they may bereevaluated. Those patients that have a disease response orstabilization from the initial dose of TPA are treated for up to sixcycles of twenty-eight days according to the protocol below.

Following the two week rest period, patients are pre-medicated withTylenol 650 mg and Benadryl 25-50 mg (depending on the patient's sizeand age) thirty minutes prior to administration of TPA. They are thengiven an intravenous infusion of TPA through a central venous catheterdaily for 5 days a week for two consecutive weeks followed by a 2-weekrest period. TPA is administered at a dose of 1 mg in 200 ml of normalsaline over 1 hour. Twenty four hours prior to initiation of TPAtherapy, patients are given 10 mg of dexamethasone every six hours and1500 mg of choline magnesium trisalicylate continuing every eight hoursuntil 24 hours after administration of the TPA.

Blood levels of TPA are measured prior to and after infusion using abioassay that measures organic solvent extractable differentiationactivity. 1 ml of blood is extracted twice with 5 ml of ethyl acetate,redissolving the extraction residue in 50 μL of ethanol and addition ofan aliquot of HL60 cells. After 48 hours, adherent cells are measured.

Tests are also run on blood samples taken prior to and after infusionwith TPA to determine levels of white blood cells, platelets, andneutrophils. The samples are additionally analyzed for the presence ofmyeloblasts and Auer rods. These and continuing experiments will furtherelucidate the therapeutic cytotoxic and other effects that TPA elicitsagainst neoplastic cells in AML and other neoplastic and malignantconditions.

Example VIII Measurement of the Modulation of ERK Activation

Phospho-ERK levels are measured in circulating malignant cells inpatients with leukemia and in peripheral blood mononuclear cells inlymphoma/solid tumor patients. A blood sample is taken from patientstreated according to the protocol of Example VII both prior to and afteradministration of TPA.

In leukemia patients with a WBC≧1000 per μL, flow cytometry is performedon a blood sample using cell surface antigen-specific and phosphor-ERKspecific antibodies directly conjugated to flurophores (BD Biosciences,San Jose, Calif.). Samples are taken pre-administration of TPA and onehour after infusion of TPA on days 1, 2 and 11 in the initial treatmentaccording to the protocol of Example VII and days 1 and 11 in subsequentcycles. In leukemia patients with an absolute leukemic blast number≧2500per μL and other non-leukemic patients, peripheral blood samples aretaken on days 1, 8 and 15 of the initial cycle according to the protocolof Example VII prior to and 1 and 4 hours post infusion. Samples arealso analyzed using Western blot analysis for phosphor-ERK, and totalERK1/2 levels to confirm the results obtained from the flow cytometryand correlated to clinical responses.

The foregoing analyses will further elucidate TPA's role in treatment ofneoplastic and malignant conditions, including TPA's cytotoxic effect onmalignant cells, exemplified by primary AML cells, and the associatedreduction by TPA of the phosphor-ERK pro-survival signal.

Example IX Measurement of NF-κB Modulation

In prior studies we have shown that NF-κB activity can be modulated inpatients following administration of TPA with dexamethasone.Additionally, dexamethasone has been shown to induce MKP-1 ERKphosphatase expression and enhance TPA cytotoxicity. The followingstudies are designed to further elucidate how NF-κB activity istherapeutically modulated in patients treated with TPA plusdexamethasone.

NF-κB binding is measured in patient peripheral blood samples atbaseline and pre and post infusion from patients treated with TPAaccording to Example VII using ELISA-based assays (BD Bioscience, SanJose, USA). NF-κB levels are quantified using chemiluminescent intensityto detect hinging in limiting amounts of cellular extract using a96-well format. Additionally, electrophoretic mobility shift assays areperformed to measure NF-κB binding in peripheral blood samples fromleukemia patient with an absolute leukemic blast number≧2500 per μL andother non-leukemic patients with normal white blood cell counts.

The foregoing studies will further PA is an inducer of NF-κB, howeverthese experiments demonstrate that AML cell NF-κB can be inhibited withtreatment with dexamethasone and choline magnesium trisalicylate.

Example X Determination of Changes in Leukemic Gene Expression

TPA induces RNA levels of several dual specificity phosphatases capableof terminating pro-survival ERK pathway signaling. A blood sample takenpre and post infusion from patients with AML treated with TPA accordingto Example VII is used to study RNA expression of AML signalingcomponents such as the MAPK-specific DUSPs using quantitative realtimeRT-PCR and oligonucleotide microarray analysis.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity and understanding, it will be apparentto the artisan that certain changes and modifications may be practicedwithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications andother references have been cited with the foregoing disclosure foreconomy of description. Each of these references is incorporated hereinby reference in its entirety for all purposes. It is noted, however,that the various publications discussed herein are incorporated solelyfor their disclosure prior to the filing date of the presentapplication, and the inventors reserve the right to antedate suchdisclosure by virtue of prior invention.

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We claim:
 1. A method for treating HIV infection or disease in amammalian subject comprising administering an effective amount of aphorbol ester of Formula I or a pharmaceutically-acceptable salt thereofto said mammalian subject

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

and R₃ is selected from hydrogen or


2. A method for treating one or more symptoms or conditions of HIVinfection or AIDS in a mammalian subject comprising administering aneffective amount of a phorbol ester of Formula I or apharmaceutically-acceptable salt thereof to said mammalian subject

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

and R₃ is selected from hydrogen or


3. A method for controlling HIV infection in a mammalian subject withAIDS comprising administering an effective amount of a phorbol ester ofFormula I or a pharmaceutically-acceptable salt thereof to saidmammalian subject

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

and R₃ is selected from hydrogen or


4. A method for activating latent reservoirs of HIV comprisingadministering an effective amount of a phorbol ester of Formula I or apharmaceutically-acceptable salt thereof to a mammalian subject in needthereof,

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

and R₃ is selected from hydrogen or


5. A method of increasing the expression of Thl cytokines comprisingadministering an effective amount of a phorbol ester of Formula I or apharmaceutically-acceptable salt thereof to a mammalian subject in needthereof,

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

and R₃ is selected from hydrogen or


6. The method according to claim 1, 2, 3, 4 or 5, wherein the phorbolester of Formula I is selected from the group consisting of phorbol13-butyrate, phorbol 12-decanoate, phorbol 13-decanoate, phorbol12,13-diacetate, phorbol 13,20-diacetate, phorbol 12,13-dibenzoate,phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate, phorbol12,13-dihexanoate, phorbol 12,13-dipropionate, phorbol 13-myristate,12-deoxyphorbol 13-angelate, 12-deoxyphorbol 13-angelate 20-acetate,12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate, 12-deoxyphorbol13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol12-tigliate 13-decanoate, 12- deoxyphorbol 13-acetate, phorbol12-acetate, phorbol-12-myristate, phorbol-12,13,20-triacetate, andphorbol 13-acetate.